U.S. patent application number 11/183015 was filed with the patent office on 2006-12-28 for cleavage of nucleic acids.
Invention is credited to Mary Ann D. Brow, James E. Dahlberg, Jeff G. Hall, Victor I. Lyamichev, James R. Prudent.
Application Number | 20060292580 11/183015 |
Document ID | / |
Family ID | 46253220 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060292580 |
Kind Code |
A1 |
Prudent; James R. ; et
al. |
December 28, 2006 |
Cleavage of nucleic acids
Abstract
The present invention relates to means for the detection and
characterization of nucleic acid sequences, as well as variations
in nucleic acid sequences. The present invention also relates to
methods for forming a nucleic acid cleavage structure on a target
sequence and cleaving the nucleic acid cleavage structure in a
site-specific manner. The structure-specific nuclease activity of a
variety of enzymes is used to cleave the target-dependent cleavage
structure, thereby indicating the presence of specific nucleic acid
sequences or specific variations thereof.
Inventors: |
Prudent; James R.; (Madison,
WI) ; Hall; Jeff G.; (Madison, WI) ;
Lyamichev; Victor I.; (Madison, WI) ; Brow; Mary Ann
D.; (Madison, WI) ; Dahlberg; James E.;
(Madison, WI) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
101 HOWARD STREET
SUITE 350
SAN FRANCISCO
CA
94105
US
|
Family ID: |
46253220 |
Appl. No.: |
11/183015 |
Filed: |
July 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09982667 |
Oct 18, 2001 |
7011944 |
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11183015 |
Jul 15, 2005 |
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09350309 |
Jul 9, 1999 |
6348314 |
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09982667 |
Oct 18, 2001 |
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08756386 |
Nov 26, 1996 |
5985557 |
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09350309 |
Jul 9, 1999 |
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08682853 |
Jul 12, 1996 |
6001567 |
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08756386 |
Nov 26, 1996 |
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08599491 |
Jan 24, 1996 |
5846717 |
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08682853 |
Jul 12, 1996 |
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Current U.S.
Class: |
435/6.1 ;
435/6.18; 536/24.3 |
Current CPC
Class: |
C12Q 1/6823 20130101;
C12Q 1/6823 20130101; Y10S 435/822 20130101; Y10S 435/81 20130101;
C12N 9/22 20130101; C12Q 1/6827 20130101; C12Q 2561/109 20130101;
C12Q 2565/525 20130101; C12Q 2563/149 20130101; C12Q 1/6827
20130101; C12Q 1/6827 20130101; C12Q 2525/301 20130101; C12Q
2563/149 20130101; C12Q 2525/301 20130101; C12Q 2561/109 20130101;
C12Q 2565/525 20130101; C12Q 2561/109 20130101; C12Q 2561/109
20130101; C12Q 2525/301 20130101; C12Q 2525/301 20130101; C12Q
2561/109 20130101; C12Q 2565/525 20130101; C12Q 1/6827 20130101;
C12Q 2561/109 20130101; C12Q 2563/149 20130101; C12Q 2561/109
20130101; C12Q 2565/525 20130101; C12Q 1/683 20130101; C12Q 1/683
20130101; C12Q 1/6823 20130101; C12Q 1/6823 20130101 |
Class at
Publication: |
435/006 ;
536/024.3 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04 |
Claims
1-25. (canceled)
26. A composition comprising a purified thermostable FEN-1
endonuclease and a polymerase.
27. The composition of claim 26, wherein said thermostable FEN-1
endonuclease comprises a FEN-1 endonuclease from an archaebacterial
species.
28. The composition of claim 27, wherein said FEN-1 endonuclease
comprises a FEN-1 endonuclease from Pyrococcus furiosus.
29. The composition of claim 26, wherein said FEN-1 endonuclease
comprises the sequence SEQ ID NO: 115.
30. The composition of claim 27, wherein said FEN-1 endonuclease
comprises a FEN-1 endonuclease from Methanococcus jannaschii.
31. The composition of claim 26, wherein said FEN-1 endonuclease
comprises the sequence SEQ ID NO: 111.
32. The composition of claim 26, wherein said thermostable FEN-1
endonuclease comprises a non-natural thermostable FEN-1
endonuclease.
33. The composition of claim 32, wherein said non-natural
thermostable FEN-1 endonuclease comprises a chimerical FEN-1
endonuclease.
34. The composition of claim 26, wherein said polymerase comprises
a thermostable polymerase.
35. The composition of claim 26, wherein said polymerase comprises
a template-independent polymerase.
36. The composition of claim 26, wherein said polymerase comprises
a template-dependent polymerase.
37. The composition of claim 26, wherein said purified thermostable
FEN-1 endonuclease and said polymerase are in a mixture.
38. The composition of claim 26, wherein said mixture comprises a
reaction mixture.
39. The composition of claim 26, further comprising a nucleic acid
molecule.
40. The composition of claim 39, wherein said nucleic acid molecule
comprises a probe oligonucleotide.
41. The composition of claim 39, wherein said nucleic acid molecule
is attached to a solid support.
42. The composition of claim 26, further comprising a buffer
comprising magnesium.
43. The composition of claim 26, further comprising a labeled
molecule.
44. The composition of claim 43, wherein said labeled molecule
comprises a labeled nucleic acid molecule.
45. The composition of claim 44, wherein said labeled nucleic acid
molecule comprises a fluorescent label.
46. The composition of claim 26, further comprising first and
second nucleic acid molecules.
47. The composition of claim 46, wherein said first and second
nucleic acid molecules are capable of hybridizing to a target
nucleic acid having a first region and a second region, said second
region downstream of and contiguous to said first region, wherein
at least a portion of said first nucleic acid molecule is
completely complementary to said first region of said target
nucleic acid and wherein said second nucleic acid molecule
comprises a 3' portion and a 5' portion, wherein said 5' portion is
completely complementary to said second region of said target
nucleic acid.
48. The composition of claim 47, further comprising a stacker
oligonucleotide.
Description
[0001] This is a Continuation-In-Part of co-pending application
Ser. No. 08/682,853, filed Jul. 12, 1996, which is a
Continuation-In-Part of co-pending application Ser. No. 08/599,491,
filed on Jan. 24, 1996.
FIELD OF THE INVENTION
[0002] The present invention relates to means for the detection and
characterization of nucleic acid sequences and variations in
nucleic acid sequences. The present invention relates to methods
for forming a nucleic acid cleavage structure on a target sequence
and cleaving the nucleic acid cleavage structure in a site-specific
manner. The 5' nuclease activity of a variety of enzymes is used to
cleave the target-dependent cleavage structure, thereby indicating
the presence of specific nucleic acid sequences or specific
variations thereof. The present invention further provides novel
methods and devices for the separation of nucleic acid molecules
based by charge.
BACKGROUND OF THE INVENTION
[0003] The detection and characterization of specific nucleic acid
sequences and sequence variations has been utilized to detect the
presence of viral or bacterial nucleic acid sequences indicative of
an infection, the presence of variants or alleles of mammalian
genes associated with disease and cancers and the identification of
the source of nucleic acids found in forensic samples, as well as
in paternity determinations.
[0004] Various methods are known to the art which may be used to
detect and characterize specific nucleic acid sequences and
sequence variants. Nonetheless, as nucleic acid sequence data of
the human genome, as well as the genomes of pathogenic organisms
accumulates, the demand for fast, reliable, cost-effective and
user-friendly tests for the detection of specific nucleic acid
sequences continues to grow. Importantly, these tests must be able
to create a detectable signal from samples which contain very few
copies of the sequence of interest. The following discussion
examines two levels of nucleic acid detection assays currently in
use: I. Signal Amplification Technology for detection of rare
sequences; and II. Direct Detection Technology for detection of
higher copy number sequences.
I. Signal Amplification Technology Methods For Amplification
[0005] The "Polymerase Chain Reaction" (PCR) comprises the first
generation of methods for nucleic acid amplification. However,
several other methods have been developed that employ the same
basis of specificity, but create signal by different amplification
mechanisms. These methods include the "Ligase Chain Reaction"
(LCR), "Self-Sustained Synthetic Reaction" (3SR/NASBA), and
"Q.beta.-Replicase" (Q.beta.).
[0006] Polymerase Chain Reaction (PCR)
[0007] The polymerase chain reaction (PCR), as described in U.S.
Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al. (the
disclosures of which are hereby incorporated by reference),
describe a method for increasing the concentration of a segment of
target sequence in a mixture of genomic DNA without cloning or
purification. This technology provides one approach to the problems
of low target sequence concentration. PCR can be used to directly
increase the concentration of the target to an easily detectable
level. This process for amplifying the target sequence involves
introducing a molar excess of two oligonucleotide primers which are
complementary to their respective strands of the double-stranded
target sequence to the DNA mixture containing the desired target
sequence. The mixture is denatured and then allowed to hybridize.
Following hybridization, the primers are extended with polymerase
so as to form complementary strands. The steps of denaturation,
hybridization, and polymerase extension can be repeated as often as
needed, in order to obtain relatively high concentrations of a
segment of the desired target sequence.
[0008] The length of the segment of the desired target sequence is
determined by the relative positions of the primers with respect to
each other, and, therefore, this length is a controllable
parameter. Because the desired segments of the target sequence
become the dominant sequences (in terms of concentration) in the
mixture, they are said to be "PCR-amplified."
[0009] Ligase Chain Reaction (LCR or LAR)
[0010] The ligase chain reaction (LCR; sometimes referred to as
"Ligase Amplification Reaction" (LAR) described by Barany, Proc.
Natl. Acad. Sci., 88:189 (1991); Barany, PCR Methods and Applic.,
1:5 (1991); and Wu and Wallace, Genomics 4:560 (1989) has developed
into a well-recognized alternative method for amplifying nucleic
acids. In LCR, four oligonucleotides, two adjacent oligonucleotides
which uniquely hybridize to one strand of target DNA, and a
complementary set of adjacent oligonucleotides, which hybridize to
the opposite strand are mixed and DNA ligase is added to the
mixture. Provided that there is complete complementarity at the
junction, ligase will covalently link each set of hybridized
molecules. Importantly, in LCR, two probes are ligated together
only when they base-pair with sequences in the target sample,
without gaps or mismatches. Repeated cycles of denaturation,
hybridization and ligation amplify a short segment of DNA. LCR has
also been used in combination with PCR to achieve enhanced
detection of single-base changes. Segev, PCT Public. No. WO9001069
A1 (1990). However, because the four oligonucleotides used in this
assay can pair to form two short ligatable fragments, there is the
potential for the generation of target-independent background
signal. The use of LCR for mutant screening is limited to the
examination of specific nucleic acid positions.
[0011] Self-Sustained Synthetic Reaction (3SR/NASBA)
[0012] The self-sustained sequence replication reaction (3SR)
(Guatelli et al., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with
an erratum at Proc. Natl. Acad. Sci., 87:7797 [1990]) is a
transcription-based in vitro amplification system (Kwok et al.,
Proc. Natl. Acad. Sci., 86:1173-1177 [1989]) that can exponentially
amplify RNA sequences at a uniform temperature. The amplified RNA
can then be utilized for mutation detection (Fahy et al., PCR Meth.
Appl., 1:25-33 [1991]). In this method, an oligonucleotide primer
is used to add a phage RNA polymerase promoter to the 5' end of the
sequence of interest. In a cocktail of enzymes and substrates that
includes a second primer, reverse transcriptase, RNase H, RNA
polymerase and ribo- and deoxyribonucleoside triphosphates, the
target sequence undergoes repeated rounds of transcription, cDNA
synthesis and second-strand synthesis to amplify the area of
interest. The use of 3SR to detect mutations is kinetically limited
to screening small segments of DNA (e.g., 200-300 base pairs).
[0013] Q-Beta (Q.beta.) Replicase
[0014] In this method, a probe which recognizes the sequence of
interest is attached to the replicatable RNA template for Q.beta.
replicase. A previously identified major problem with false
positives resulting from the replication of unhybridized probes has
been addressed through use of a sequence-specific ligation step.
However, available thermostable DNA ligases are not effective on
this RNA substrate, so the ligation must be performed by T4 DNA
ligase at low temperatures (37.degree. C.). This prevents the use
of high temperature as a means of achieving specificity as in the
LCR, the ligation event can be used to detect a mutation at the
junction site, but not elsewhere.
[0015] Table 1 below, lists some of the features desirable for
systems useful in sensitive nucleic acid diagnostics, and
summarizes the abilities of each of the major amplification methods
(See also, Landgren, Trends in Genetics 9:199 [1993]).
[0016] A successful diagnostic method must be very specific. A
straight-forward method of controlling the specificity of nucleic
acid hybridization is by controlling the temperature of the
reaction. While the 3SR/NASBA, and Q.beta. systems are all able to
generate a large quantity of signal, one or more of the enzymes
involved in each cannot be used at high temperature (i.e.,
>55.degree. C.). Therefore the reaction temperatures cannot be
raised to prevent non-specific hybridization of the probes. If
probes are shortened in order to make them melt more easily at low
temperatures, the likelihood of having more than one perfect match
in a complex genome increases. For these reasons, PCR and LCR
currently dominate the research field in detection technologies.
TABLE-US-00001 TABLE 1 METHOD: PCR & 3SR FEATURE PCR LCR LCR
NASBA Q.beta. Amplifies Target + + + + Recognition of Independent +
+ + + + Sequences Required Performed at High Temp. + + Operates at
Fixed Temp. + + Exponential Amplification + + + + + Generic Signal
Generation + Easily Automatable
[0017] The basis of the amplification procedure in the PCR and LCR
is the fact that the products of one cycle become usable templates
in all subsequent cycles, consequently doubling the population with
each cycle. The final yield of any such doubling system can be
expressed as: (1+X).sup.n=y, where "X" is the mean efficiency
(percent copied in each cycle), "n" is the number of cycles, and
"y" is the overall efficiency, or yield of the reaction (Mullis,
PCR Methods Applic., 1:1 [1991]). If every copy of a target DNA is
utilized as a template in every cycle of a polymerase chain
reaction, then the mean efficiency is 100%. If 20 cycles of PCR are
performed, then the yield will be 2.sup.20, or 1,048,576 copies of
the starting material. If the reaction conditions reduce the mean
efficiency to 85%, then the yield in those 20 cycles will be only
1.85.sup.20, or 220,513 copies of the starting material. In other
words, a PCR running at 85% efficiency will yield only 21% as much
final product, compared to a reaction running at 100% efficiency. A
reaction that is reduced to 50% mean efficiency will yield less
than 1% of the possible product.
[0018] In practice, routine polymerase chain reactions rarely
achieve the theoretical maximum yield, and PCRs are usually run for
more than 20 cycles to compensate for the lower yield. At 50% mean
efficiency, it would take 34 cycles to achieve the million-fold
amplification theoretically possible in 20, and at lower
efficiencies, the number of cycles required becomes prohibitive. In
addition, any background products that amplify with a better mean
efficiency than the intended target will become the dominant
products.
[0019] Also, many variables can influence the mean efficiency of
PCR, including target DNA length and secondary structure, primer
length and design, primer and dNTP concentrations, and buffer
composition, to name but a few. Contamination of the reaction with
exogenous DNA (e.g., DNA spilled onto lab surfaces) or
cross-contamination is also a major consideration. Reaction
conditions must be carefully optimized for each different primer
pair and target sequence, and the process can take days, even for
an experienced investigator. The laboriousness of this process,
including numerous technical considerations and other factors,
presents a significant drawback to using PCR in the clinical
setting. Indeed, PCR has yet to penetrate the clinical market in a
significant way. The same concerns arise with LCR, as LCR must also
be optimized to use different oligonucleotide sequences for each
target sequence. In addition, both methods require expensive
equipment, capable of precise temperature cycling.
[0020] Many applications of nucleic acid detection technologies,
such as in studies of allelic variation, involve not only detection
of a specific sequence in a complex background, but also the
discrimination between sequences with few, or single, nucleotide
differences. One method for the detection of allele-specific
variants by PCR is based upon the fact that it is difficult for Taq
polymerase to synthesize a DNA strand when there is a mismatch
between the template strand and the 3' end of the primer. An
allele-specific variant may be detected by the use of a primer that
is perfectly matched with only one of the possible alleles; the
mismatch to the other allele acts to prevent the extension of the
primer, thereby preventing the amplification of that sequence. This
method has a substantial limitation in that the base composition of
the mismatch influences the ability to prevent extension across the
mismatch, and certain mismatches do not prevent extension or have
only a minimal effect (Kwok et al., Nucl. Acids Res., 18:999
[1990]).)
[0021] A similar 3'-mismatch strategy is used with greater effect
to prevent ligation in the LCR (Barany, PCR Meth. Applic., 1:5
[1991]). Any mismatch effectively blocks the action of the
thermostable ligase, but LCR still has the drawback of
target-independent background ligation products initiating the
amplification. Moreover, the combination of PCR with subsequent LCR
to identify the nucleotides at individual positions is also a
clearly cumbersome proposition for the clinical laboratory.
II. Direct Detection Technology
[0022] When a sufficient amount of a nucleic acid to be detected is
available, there are advantages to detecting that sequence
directly, instead of making more copies of that target, (e.g., as
in PCR and LCR). Most notably, a method that does not amplify the
signal exponentially is more amenable to quantitative analysis.
Even if the signal is enhanced by attaching multiple dyes to a
single oligonucleotide, the correlation between the final signal
intensity and amount of target is direct. Such a system has an
additional advantage that the products of the reaction will not
themselves promote further reaction, so contamination of lab
surfaces by the products is not as much of a concern. Traditional
methods of direct detection including Northern and Southern
blotting and RNase protection assays usually require the use of
radioactivity and are not amenable to automation. Recently devised
techniques have sought to eliminate the use of radioactivity and/or
improve the sensitivity in automatable formats. Two examples are
the "Cycling Probe Reaction" (CPR), and "Branched DNA" (bDNA)
[0023] The cycling probe reaction (CPR) (Duck et al., BioTech.,
9:142 [1990]), uses a long chimeric oligonucleotide in which a
central portion is made of RNA while the two termini are made of
DNA. Hybridization of the probe to a target DNA and exposure to a
thermostable RNase H causes the RNA portion to be digested. This
destabilizes the remaining DNA portions of the duplex, releasing
the remainder of the probe from the target DNA and allowing another
probe molecule to repeat the process. The signal, in the form of
cleaved probe molecules, accumulates at a linear rate. While the
repeating process increases the signal, the RNA portion of the
oligonucleotide is vulnerable to RNases that may be carried through
sample preparation.
[0024] Branched DNA (bDNA), described by Urdea et al., Gene
61:253-264 (1987), involves oligonucleotides with branched
structures that allow each individual oligonucleotide to carry 35
to 40 labels (e.g., alkaline phosphatase enzymes). While this
enhances the signal from a hybridization event, signal from
non-specific binding is similarly increased.
[0025] While both of these methods have the advantages of direct
detection discussed above, neither the CPR or bDNA methods can make
use of the specificity allowed by the requirement of independent
recognition by two or more probe (oligonucleotide) sequences, as is
common in the signal amplification methods described in section I.
above. The requirement that two oligonucleotides must hybridize to
a target nucleic acid in order for a detectable signal to be
generated confers an extra measure of stringency on any detection
assay. Requiring two oligonucleotides to bind to a target nucleic
acid reduces the chance that false "positive" results will be
produced due to the non-specific binding of a probe to the target.
The further requirement that the two oligonucleotides must bind in
a specific orientation relative to the target, as is required in
PCR, where oligonucleotides must be oppositely but appropriately
oriented such that the DNA polymerase can bridge the gap between
the two oligonucleotides in both directions, further enhances
specificity of the detection reaction. However, it is well known to
those in the art that even though PCR utilizes two oligonucleotide
probes (termed primers) "non-specific" amplification (ie.,
amplification of sequences not directed by the two primers used) is
a common artifact. This is in part because the DNA polymerase used
in PCR can accommodate very large distances, measured in
nucleotides, between the oligonucleotides and thus there is a large
window in which non-specific binding of an oligonucleotide can lead
to exponential amplification of inappropriate product. The LCR, in
contrast, cannot proceed unless the oligonucleotides used are bound
to the target adjacent to each other and so the full benefit of the
dual oligonucleotide hybridization is realized.
[0026] An ideal direct detection method would combine the
advantages of the direct detection assays (e.g., easy
quantification and minimal risk of carry-over contamination) with
the specificity provided by a dual oligonucleotide hybridization
assay.
SUMMARY OF THE INVENTION
[0027] The present invention relates to means for cleaving a
nucleic acid cleavage structure in a site-specific manner. In one
embodiment, the means for cleaving is a cleaving enzyme comprising
5' nucleases derived from thermostable DNA polymerases. These
polymerases form the basis of a novel method of detection of
specific nucleic acid sequences. The present invention contemplates
use of novel detection methods for various uses, including, but not
limited to clinical diagnostic purposes.
[0028] In one embodiment, the present invention contemplates a DNA
sequence encoding a DNA polymerase altered in sequence (i.e., a
"mutant" DNA polymerase) relative to the native sequence, such that
it exhibits altered DNA synthetic activity from that of the native
(i.e., "wild type") DNA polymerase. It is preferred that the
encoded DNA polymerase is altered such that it exhibits reduced
synthetic activity compared to that of the native DNA polymerase.
In this manner, the enzymes of the invention are predominantly 5'
nucleases and are capable of cleaving nucleic acids in a
structure-specific manner in the absence of interfering synthetic
activity.
[0029] Importantly, the 5' nucleases of the present invention are
capable of cleaving linear duplex structures to create single
discrete cleavage products. These linear structures are either 1)
not cleaved by the wild type enzymes (to any significant degree),
or 2) are cleaved by the wild type enzymes so as to create multiple
products. This characteristic of the 5' nucleases has been found to
be a consistent property of enzymes derived in this manner from
thermostable polymerases across eubacterial thermophilic
species.
[0030] It is not intended that the invention be limited by the
nature of the alteration necessary to render the polymerase
synthesis-deficient. Nor is it intended that the invention be
limited by the extent of the deficiency. The present invention
contemplates various structures, including altered structures
(primary, secondary, etc.), as well as native structures, that may
be inhibited by synthesis inhibitors.
[0031] Where the polymerase structure is altered, it is not
intended that the invention be limited by the means by which the
structure is altered. In one embodiment, the alteration of the
native DNA sequence comprises a change in a single nucleotide. In
another embodiment, the alteration of the native DNA sequence
comprises a deletion of one or more nucleotides. In yet another
embodiment, the alteration of the native DNA sequence comprises an
insertion of one or more nucleotides. It is contemplated that the
change in DNA sequence may manifest itself as change in amino acid
sequence.
[0032] The present invention contemplates structure-specific
nucleases from a variety of sources, including mesophilic,
psychrophilic, thermophilic, and hyperthermophilic organisms. The
preferred structure-specific nucleases are thermostable.
Thermostable structure-specific nucleases are contemplated as
particularly useful in that they operate at temperatures where
nucleic acid hybridization is extremely specific, allowing for
allele-specific detection (including single-base mismatches). In
one embodiment, the thermostable structure-specific are
thermostable 5' nucleases which are selected from the group
consisting of altered polymerases derived from the native
polymerases of Thermus species, including, but not limited to
Thermus aquaticus, Thermus flavus, and Thermus thermophilus.
However, the invention is not limited to the use of thermostable 5'
nucleases. Thermostable structure-specific nucleases from the
FEN-1, RAD2 and XPG class of nucleases are also preferred.
[0033] The present invention provides a composition comprising a
cleavage structure, said cleavage structure comprising: a) a target
nucleic acid, said target nucleic acid having a first region, a
second region, a third region and a fourth region, wherein said
first region is located adjacent to and downstream from said second
region, said second region is located adjacent to and downstream
from said third region and said third region is located adjacent to
and downstream from said fourth region; b) a first oligonucleotide
complementary to said fourth region of said target nucleic acid; c)
a second oligonucleotide having a 5' portion and a 3' portion
wherein said 5' portion of said second oligonucleotide contains a
sequence complementary to said second region of said target nucleic
acid and wherein said 3' portion of said second oligonucleotide
contains a sequence complementary to said third region of said
target nucleic acid; and d) a third oligonucleotide having a 5'
portion and a 3' portion wherein said 5' portion of said third
oligonucleotide contains a sequence complementary to said first
region of said target nucleic acid and wherein said 3' portion of
said third oligonucleotide contains a sequence complementary to
said second region of said target nucleic acid.
[0034] The present invention is not limited by the length of the
four regions of the target nucleic acid. In one embodiment, the
first region of the target nucleic acid has a length of 11 to 50
nucleotides. In another embodiment, the second region of the target
nucleic acid has a length of one to three nucleotides. In another
embodiment, the third region of the target nucleic acid has a
length of six to nine nucleotides. In yet another embodiment, the
fourth region of the target nucleic acid has a length of 6 to 50
nucleotides.
[0035] The invention is not limited by the nature or composition of
the of the first, second, third and fourth oligonucleotides; these
oligonucleotides may comprise DNA, RNA, PNA and combinations
thereof as well as comprise modified nucleotides, universal bases,
adducts, etc. Further, one or more of the first, second, third and
the fourth oligonucleotides may contain a dideoxynucleotide at the
3' terminus.
[0036] In a preferred embodiment, the target nucleic acid is not
completely complementary to at least one of the first, the second,
the third and the fourth oligonucleotides. In a particularly
preferred embodiment, the target nucleic acid is not completely
complementary to the second oligonucleotide.
[0037] As noted above, the present invention contemplates the use
of structure-specific nucleases in a detection method. In one
embodiment, the present invention provides a method of of detecting
the presence of a target nucleic acid molecule by detecting
non-target cleavage products comprising: a) providing: i) a
cleavage means, ii) a source of target nucleic acid, the target
nucleic acid having a first region, a second region, a third region
and a fourth region, wherein the first region is located adjacent
to and downstream from the second region, the second region is
located adjacent to and downstream from the third region and the
third region is located adjacent to and downstream from the fourth
region; iii) a first oligonucleotide complementary to the fourth
region of the target nucleic acid; iv) a second oligonucleotide
having a 5' portion and a 3' portion wherein the 5' portion of the
second oligonucleotide contains a sequence complementary to the
second region of said target nucleic acid and wherein the 3'
portion of the second oligonucleotide contains a sequence
complementary to the third region of the target nucleic acid; iv) a
third oligonucleotide having a 5' and a 3' portion wherein the 5'
portion of the third oligonucleotide contains a sequence
complementary to the first region of the target nucleic acid and
wherein the 3' portion of the third oligonucleotide contains a
sequence complementary to the second region of the target nucleic
acid; b) mixing the cleavage means, the target nucleic acid, the
first oligonucleotide, the second oligonucleotide and the third
oligonucleotide to create a reaction mixture under reaction
conditions such that the first oligonucleotide is annealed to the
fourth region of the target nucleic acid and wherein at least the
3' portion of the second oligonucleotide is annealed to the target
nucleic acid and wherein at least the 5' portion of the third
oligonucleotide is annealed to the target nucleic acid so as to
create a cleavage structure and wherein cleavage of the cleavage
structure occurs to generate non-target cleavage products, each
non-target cleavage product having a 3'-hydroxyl group; and c)
detecting the non-target cleavage products.
[0038] The invention is not limited by the nature of the target
nucleic acid. In one embodiment, the target nucleic acid comprises
single-stranded DNA. In another embodiment, the target nucleic acid
comprises double-stranded DNA and prior to step c), the reaction
mixture is treated such that the double-stranded DNA is rendered
substantially single-stranded. In another embodiment, the target
nucleic acid comprises RNA and the first and second
oligonucleotides comprise DNA.
[0039] The invention is not limited by the nature of the cleavage
means. In one embodiment, the cleavage means is a
structure-specific nuclease; particularly preferred
structure-specific nucleases are thermostable structure-specific
nucleases. In a preferrred embodiment, the thermostable
structure-specific nuclease is encoded by a DNA sequence selected
from the group consisting of SEQ ID NOS:1-3, 9, 10, 12, 21, 30, and
31.
[0040] In a preferred embodiment, the detection of the non-target
cleavage products comprises electrophoretic separation of the
products of the reaction followed by visualization of the separated
non-target cleavage products.
[0041] In another preferred embodiment, one or more of the first,
second, and third oligonucleotides contain a dideoxynucleotide at
the 3' terminus. When dideoxynucleotide-containing oligonucleotides
are employed, the detection of the non-target cleavage products
preferably comprises: a) incubating said non-target cleavage
products With a template-independent polymerase and at least one
labelled nucleoside triphosphate under conditions such that at
least one labelled nucleotide is added to the 3'-hydroxyl group of
said non-target cleavage products to generate labelled non-target
cleavage products; and b) detecting the presence of said labelled
non-target cleavage products. The invention is not limited by the
nature of the template-independent polymerase employed; in one
embodiment, the template-independent polymerase is selected from
the group consisting of terminal deoxynucleotidyl transferase (TdT)
and poly A polymerase. When TdT or polyA polymerase are employed in
the detection step, the second oligonucleotide may contain a 5' end
label, the 5' end label being a different label than the label
present upon the labelled nucleoside triphosphate. The inevntion is
not limited by the nature of the 5' end label; a wide variety of
suitable 5' end labels are known to the art and include biotin,
fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3
amidite, Cy5 amidite and digoxigenin.
[0042] In another embodiment, detecting the non-target cleavage
products comprises: a) incubating said non-target cleavage products
with a template-independent polymerase and at least one nucleoside
triphosphate under conditions such that at least one nucleotide is
added to the 3'-hydroxyl group of the non-target cleavage products
to generate tailed non-target cleavage products; and b) detecting
the presence of the tailed non-target cleavage products. The
invention is not limited by the nature of the template-independent
polymerase employed; in one embodiment, the template-independent
polymerase is selected from the group consisting of terminal
deoxynucleotidyl transferase (TdT) and poly A polymerase. When TdT
or polyA polymerase are employed in the detection step, the second
oligonucleotide may contain a 5' end label. The inevntion is not
limited by the nature of the 5' end label; a wide variety of
suitable 5' end labels are known to the art and include biotin,
fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3
amidite, Cy5 amidite and digoxigenin.
[0043] In a preferred embodiment, the reaction conditions comprise
providing a source of divalent cations; particularly preferred
divalent cations are Mn.sup.2+ and Mg.sup.2+ ions.
[0044] The present invention further provides a method of detecting
the presence of a target nucleic acid molecule by detecting
non-target cleavage products comprising: a) providing: i) a
cleavage means, ii) a source of target nucleic acid, said target
nucleic acid having a first region, a second region and a third
region, wherein said first region is located adjacent to and
downstream from said second region and wherein said second region
is located adjacent to and downstream from said third region; iii)
a first oligonucleotide having a 5' and a 3' portion wherein said
5' portion of said first oligonucleotide contains a sequence
complementary to said second region of said target nucleic acid and
wherein said 3' portion of said first oligonucleotide contains a
sequence complementary to said third region of said target nucleic
acid; iv) a second oligonucleotide having a length between eleven
to fifteen nucleotides and further having a 5' and a 3' portion
wherein said 5' portion of said second oligonucleotide contains a
sequence complementary to said first region of said target nucleic
acid and wherein said 3' portion of said second oligonucleotide
contains a sequence complementary to said second region of said
target nucleic acid; b) mixing said cleavage means, said target
nucleic acid, said first oligonucleotide and said second
oligonucleotide to create a reaction mixture under reaction
conditions such that at least said 3' portion of said first
oligonucleotide is annealed to said target nucleic acid and wherein
at least said 5' portion of said second oligonucleotide is annealed
to said target nucleic acid so as to create a cleavage structure
and wherein cleavage of said cleavage structure occurs to generate
non-target cleavage products, each non-target cleavage product
having a 3'-hydroxyl group; and c) detecting said non-target
cleavage products. In a preferred embodiment the cleavage means is
a structure-specific nuclease, preferably a thermostable
structure-specific nuclease.
[0045] The invention is not limited by the length of the various
regions of the target nucleic acid. In a preferred embodiment, the
second region of said target nucleic acid has a length between one
to five nucleotides. In another preferred embodiment, one or more
of the first and the second oligonucleotides contain a
dideoxynucleotide at the 3' terminus. When
dideoxynucleotide-containing oligonucleotides are employed, the
detection of the non-target cleavage products preferably comprises:
a) incubating said non-target cleavage products with a
template-independent polymerase and at least one labelled
nucleoside triphosphate under conditions such that at least one
labelled nucleotide is added to the 3'-hydroxyl group of said
non-target cleavage products to generate labelled non-target
cleavage products; and b) detecting the presence of said labelled
non-target cleavage products. The invention is not limited by the
nature of the template-independent polymerase employed; in one
embodiment, the template-independent polymerase is selected from
the group consisting of terminal deoxynucleotidyl transferase (TdT)
and poly A polymerase. When TdT or polyA polymerase are employed in
the detection step, the second oligonucleotide may contain a 5' end
label, the 5' end label being a different label than the label
present upon the labelled nucleoside triphosphate. The inevntion is
not limited by the nature of the 5' end label; a wide variety of
suitable 5' end labels are known to the art and include biotin,
fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3
amidite, Cy5 amidite and digoxigenin.
[0046] In another embodiment, detecting the non-target cleavage
products comprises: a) incubating said non-target cleavage products
with a template-independent polymerase and at least one nucleoside
triphosphate under conditions such that at least one nucleotide is
added to the 3'-hydroxyl group of the non-target cleavage products
to generate tailed non-target cleavage products; and b) detecting
the presence of the tailed non-target cleavage products. The
invention is not limited by the nature of the template-independent
polymerase employed; in one embodiment, the template-independent
polymerase is selected from the group consisting of terminal
deoxynucleotidyl transferase (TdT) and poly A polymerase. When TdT
or polyA polymerase are employed in the detection step, the second
oligonucleotide may contain a 5' end label. The inevntion is not
limited by the nature of the 5' end label; a wide variety of
suitable 5' end labels are known to the art and include biotin,
fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3
amidite, Cy5 amidite and digoxigenin.
[0047] The novel detection methods of the invention may be employed
for the detection of target DNAs and RNAs including, but not
limited to, target DNAs and RNAs comprising wild type and mutant
alleles of genes, including genes from humans or other animals that
are or may be associated with disease or cancer. In addition, the
methods of the invention may be used for the detection of and/or
identification of strains of microorganisms, including bacteria,
fungi, protozoa, ciliates and viruses (and in particular for the
detection and identification of RNA viruses, such as HCV).
DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1A provides a schematic of one embodiment of the
detection method of the present invention.
[0049] FIG. 1B provides a schematic of a second embodiment of the
detection method of the present invention.
[0050] FIG. 2 is a comparison of the nucleotide structure of the
DNAP genes isolated from Thermus aquaticus (SEQ ID NO: 1), Thermus
flavus (SEQ ID NO:2) and Thermus thermophilus (SEQ ID NO:3); the
consensus sequence (SEQ ID NO:7) is shown at the top of each
row.
[0051] FIG. 3 is a comparison of the amino acid sequence of the
DNAP isolated from Thermus aquaticus (SEQ ID NO:4), Thermus flavus
(SEQ ID NO:5), and Thermus thermophilus (SEQ ID NO:6); the
consensus sequence (SEQ ID NO:8) is shown at the top of each
row.
[0052] FIGS. 4A-G are a set of diagrams of wild-type and
synthesis-deficient DNAPTaq genes.
[0053] FIG. 5A depicts the wild-type Thermus flavus polymerase
gene.
[0054] FIG. 5B depicts a synthesis-deficient Thermus flavus
polymerase gene.
[0055] FIG. 6 depicts a structure which cannot be amplified using
DNAPTaq.
[0056] FIG. 7 is a ethidium bromide-stained gel demonstrating
attempts to amplify a bifurcated duplex using either DNAPTaq or
DNAPStf (i.e., the Stoffel fragment of DNAPTaq).
[0057] FIG. 8 is an autoradiogram of a gel analyzing the cleavage
of a bifurcated duplex by DNAPTaq and lack of cleavage by
DNAPStf.
[0058] FIGS. 9A-B are a set of autoradiograms of gels analyzing
cleavage or lack of cleavage upon addition of different reaction
components and change of incubation temperature during attempts to
cleave a bifurcated duplex with DNAPTaq.
[0059] FIGS. 10A-B are an autoradiogram displaying timed cleavage
reactions, with and without primer.
[0060] FIGS. 11A-B are a set of autoradiograms of gels
demonstrating attempts to cleave a bifurcated duplex (with and
without primer) with various DNAPs.
[0061] FIG. 12A shows the substrates and oligonucleotides used to
test the specific cleavage of substrate DNAs targeted by pilot
oligonucleotides.
[0062] FIG. 12B shows an autoradiogram of a gel showing the results
of cleavage reactions using the substrates and oligonucleotides
shown FIG. 12A.
[0063] FIG. 13A shows the substrate and oligonucleotide used to
test the specific cleavage of a substrate RNA targeted by a pilot
oligonucleotide.
[0064] FIG. 13B shows an autoradiogram of a gel showing the results
of a cleavage reaction using the substrate and oligonucleotide
shown in FIG. 13A.
[0065] FIG. 14 is a diagram of vector pTTQ18.
[0066] FIG. 15 is a diagram of vector pET-3c.
[0067] FIG. 16A-E depicts a set of molecules which are suitable
substrates for cleavage by the 5' nuclease activity of DNAPs.
[0068] FIG. 17 is an autoradiogram of a gel showing the results of
a cleavage reaction run with synthesis-deficient DNAPs.
[0069] FIG. 18 is an autoradiogram of a PEI chromatogram resolving
the products of an assay for synthetic activity in
synthesis-deficient DNAPTaq clones.
[0070] FIG. 19A depicts the substrate molecule used to test the
ability of synthesis-deficient DNAPs to cleave short hairpin
structures.
[0071] FIG. 19B shows an autoradiogram of a gel resolving the
products of a cleavage reaction run using the substrate shown in
FIG. 19A.
[0072] FIG. 20A shows the A- and T-hairpin molecules used in the
trigger/detection assay.
[0073] FIG. 20B shows the sequence of the alpha primer used in the
trigger/detection assay.
[0074] FIG. 20C shows the structure of the cleaved A- and T-hairpin
molecules.
[0075] FIG. 20D depicts the complementarity between the A- and
T-hairpin molecules.
[0076] FIG. 21 provides the complete 206-mer duplex sequence
employed as a substrate for the 5' nucleases of the present
invention FIGS. 22A and B show the cleavage of linear nucleic acid
substrates (based on the 206-mer of FIG. 21) by wild type DNAPs and
5' nucleases isolated from Thermus aquaticus and Thermus
flavus.
[0077] FIG. 23 provides a detailed schematic corresponding to the
of one embodiment of the detection method of the present
invention.
[0078] FIG. 24 shows the propagation of cleavage of the linear
duplex nucleic acid structures of FIG. 23 by the 5' nucleases of
the present invention.
[0079] FIG. 25A shows the "nibbling" phenomenon detected with the
DNAPs of the present invention.
[0080] FIG. 25B shows that the "nibbling" of FIG. 25A is 5'
nucleolytic cleavage and not phosphatase cleavage.
[0081] FIG. 26 demonstrates that the "nibbling" phenomenon is
duplex dependent.
[0082] FIG. 27 is a schematic showing how "nibbling" can be
employed in a detection assay.
[0083] FIG. 28 demonstrates that "nibbling" can be target
directed.
[0084] FIG. 29 provides a schematic drawing of a target nucleic
acid with an invader oligonucleotide and a probe oligonucleotide
annealed to the target.
[0085] FIG. 30 provides a schematic showing the S-60 hairpin
oligonucleotide (SEQ ID NO:40) with the annealed P-15
oligonucletide (SEQ ID NO:41).
[0086] FIG. 31 is an autoradiogram of a gel showing the results of
a cleavage reaction run using the S-60 hairpin in the presence or
absence of the P-15 oligonucleotide.
[0087] FIG. 32 provides a schematic showing three different
arrangements of target-specific oligonucleotides and their
hybridization to a target nucleic acid which also has a probe
oligonucleotide annealed thereto.
[0088] FIG. 33 is the image generated by a fluorescence imager
showing that the presence of an invader oligonucleotide causes a
shift in the site of cleavage in a probe/target duplex.
[0089] FIG. 34 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run using
the three target-specific oligonucleotides diagrammed in FIG.
32.
[0090] FIG. 35 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence or absence of non-target nucleic acid molecules.
[0091] FIG. 36 is the image generated by a fluoroscence imager
showing the products of invader-directed cleavage assays run in the
presence of decreasing amounts of target nucleic acid.
[0092] FIG. 37 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence or absence of saliva extract using various thermostable 5'
nucleases or DNA polymerases.
[0093] FIG. 38 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run using
various 5' nucleases.
[0094] FIG. 39 is the image generated by a fluoroscence imager
showing the products of invader-directed cleavage assays run using
two target nucleic acids which differ by a single basepair at two
different reaction temperatures.
[0095] FIG. 40A provides a schematic showing the effect of elevated
temperature upon the annealing and cleavage of a probe
oligonucleotide along a target nucleic acid wherein the probe
contains a region of noncomplementarity with the target.
[0096] FIG. 40B provides a schematic showing the effect of adding
an upstream oligonucleotide upon the annealing and cleavage of a
probe oligonucleotide along a target nucleic acid wherein the probe
contains a region of noncomplementarity with the target.
[0097] FIG. 41 provides a schematic showing an arrangement of a
target-specific invader oligonucleotide (SEQ ID NO:50) and a
target-specific probe oligonucleotide (SEQ ID NO:49) bearing a 5'
Cy3 label along a target nucleic acid (SEQ ID NO:42).
[0098] FIG. 42 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing concentrations of KCl.
[0099] FIG. 43 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing concentrations of NaCl.
[0100] FIG. 44 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing concentrations of LiCl.
[0101] FIG. 45 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing concentrations of KGlu.
[0102] FIG. 46 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing concentrations of MnCl.sub.2 or
MgCl.sub.2.
[0103] FIG. 47 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing concentrations of CTAB.
[0104] FIG. 48 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing concentrations of PEG.
[0105] FIG. 49 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of glycerol, Tween-20 and/or Nonidet-P40.
[0106] FIG. 50 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing concentrations of gelatin in reactions
containing or lacking KCl or LiCl.
[0107] FIG. 51 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run in the
presence of increasing amounts of genomic DNA or tRNA.
[0108] FIG. 52 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run use a
HCV RNA target.
[0109] FIG. 53 is the image generated by a fluorescence imager
showing the products of invader-directed cleavage assays run using
a HCV RNA target and demonstrate the stability of RNA targets under
invader-directed cleavage assay conditions.
[0110] FIG. 54 is the image generated by a fluorescence imager
showing the sensitivity of detection and the stability of RNA in
invader-directed cleavage assays run using a HCV RNA target.
[0111] FIG. 55 is the image generated by a fluorescence imager
showing thermal degradation of oligonucleotides containing or
lacking a 3' phosphate group.
[0112] FIG. 56 depicts the structure of amino-modified
oligonucleotides 70 and 74.
[0113] FIG. 57 depicts the structure of amino-modified
oligonucleotide 75 FIG. 58 depicts the structure of amino-modified
oligonucleotide 76.
[0114] FIG. 59 is the image generated by a fluorescence imager scan
of an IEF gel showing the migration of substrates 70, 70dp, 74,
74dp, 75, 75dp, 76 and 76dp.
[0115] FIG. 60A provides a schematic showing an arrangement of a
target-specific invader oligonucleotide (SEQ ID NO:61) and a
target-specific probe oligonucleotide (SEQ ID NO:62) bearing a 5'
Cy3 label along a target nucleic acid (SEQ ID NO:63).
[0116] FIG. 60B is the image generated by a fluorescence imager
showing the detection of specific cleavage products generated in an
invasive cleavage assay using charge reversal (i.e., charge based
separation of cleavage products).
[0117] FIG. 61 is the image generated by a fluorescence imager
which depicts the sensitivity of detection of specific cleavage
products generated in an invasive cleavage assay using charge
reversal.
[0118] FIG. 62 depicts a first embodiment of a device for the
charge-based separation of oligonucleotides.
[0119] FIG. 63 depicts a second embodiment of a device for the
charge-based separation of oligonucleotides.
[0120] FIG. 64 shows an autoradiogram of a gel showing the results
of cleavage reactions run in the presence or absence of a primer
oligonucleotide; a sequencing ladder is shown as a size marker.
[0121] FIGS. 65a-d depict four pairs of oligonucleotides; in each
pair shown, the upper arrangement of a probe annealed to a target
nucleic acid lacks an upstream oligonucleotide and the lower
arrangement contains an upstream oligonucleotide.
[0122] FIG. 66 shows the chemical structure of several positively
charged heterodimeric DNA-binding dyes.
[0123] FIG. 67 is a schematic showing alternative methods for the
tailing and detection of specific cleavage products in the context
of the Invader.TM.-directed cleavage assay.
[0124] FIG. 68 provides a schematic drawing of a target nucleic
acid with an Invader.TM. oligonucleotide, a miniprobe, and a
stacker oligonucleotide annealed to the target.
[0125] FIG. 69 provides a space-filling model of the 3-dimensional
structure of the T5 5'-exonuclease.
[0126] FIG. 70 provides an alignment of the amino acid sequences of
several FEN-1 nucleases including the Methanococcus jannaschii
FEN-1 protein (MJAFEN1.PRO), the Pyrococcus furiosus FEN-1 protein
(PFUFEN1.PRO), the human FEN-1 protein (HUMFEN1.PRO), the mouse
FEN-1 protein (MUSFEN1.PRO), the Saccharomyces cerevisiae YKL510
protein (YST510.PRO), the Saccharomyces cerevisiae RAD2 protein
(YSTRAD2.PRO), the Shizosaccharomyces pombe RAD13 protein
(SPORAD13.PRO), the human XPG protein (HUMXPG.PRO), the mouse XPG
protein (MUSXPG.PRO), the Xenopus laevis XPG protein (XENXPG.PRO)
and the C. elegans RAD2 protein (CELRAD2.PRO); portions of the
amino acid sequence of some of these proteins were not shown in
order to maximize the alignment between proteins. The numbers to
the left of each line of sequence refers to the amino acid residue
number; dashes represent gaps introduced to maximize alignment.
[0127] FIG. 71 provides a schematic showing the S-33 and 11-8-0
oligonucleotides in a folded configuration; the cleavage site is
indicated by the arrowhead.
DEFINITIONS
[0128] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides such as an oligonucleotide or a target
nucleic acid) related by the base-pairing rules. For example, for
the sequence "A-G-T," is complementary to the sequence "T-C-A."
Complementarity may be "partial," in which only some of the nucleic
acids' bases are matched according to the base pairing rules. Or,
there may be "complete" or "total" complementarity between the
nucleic acids. The degree of complementarity between nucleic acid
strands has significant effects on the efficiency and strength of
hybridization between nucleic acid strands. This is of particular
importance in amplification reactions, as well as detection methods
which depend upon binding between nucleic acids.
[0129] The term "homology" refers to a degree of identity. There
may be partial homology or complete homology. A partially identical
sequence is one that is less than 100% identical to another
sequence.
[0130] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the T.sub.m of the formed
hybrid, and the G:C ratio within the nucleic acids.
[0131] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation:
T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization (1985). Other
references include more sophisticated computations which take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0132] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds, under which nucleic acid hybridizations are
conducted. With "high stringency" conditions, nucleic acid base
pairing will occur only between nucleic acid fragments that have a
high frequency of complementary base sequences. Thus, conditions of
"weak" or "low" stringency are often required when it is desired
that nucleic acids which are not completely complementary to one
another be hybridized or annealed together.
[0133] The term "gene" refers to a DNA sequence that comprises
control and coding sequences necessary for the production of a
polypeptide or precursor. The polypeptide can be encoded by a full
length coding sequence or by any portion of the coding sequence so
long as the desired enzymatic activity is retained.
[0134] The term "wild-type" refers to a gene or gene product which
has the characteristics of that gene or gene product when isolated
from a naturally occurring source. A wild-type gene is that which
is most frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene. In contrast,
the term "modified" or "mutant" refers to a gene or gene product
which displays modifications in sequence and or functional
properties (i.e., altered characteristics) when compared to the
wild-type gene or gene product. It is noted that
naturally-occurring mutants can be isolated; these are identified
by the fact that they have altered characteristics when compared to
the wild-type gene or gene product.
[0135] The term "recombinant DNA vector" as used herein refers to
DNA sequences containing a desired coding sequence and appropriate
DNA sequences necessary for the expression of the operably linked
coding sequence in a particular host organism. DNA sequences
necessary for expression in procaryotes include a promoter,
optionally an operator sequence, a ribosome binding site and
possibly other sequences. Eukaryotic cells are known to utilize
promoters, polyadenlyation signals and enhancers.
[0136] The term "LTR" as used herein refers to the long terminal
repeat found at each end of a provirus (i.e., the integrated form
of a retrovirus). The LTR contains numerous regulatory signals
including transcriptional control elements, polyadenylation signals
and sequences needed for replication and integration of the viral
genome. The viral LTR is divided into three regions called U3, R
and US.
[0137] The U3 region contains the enhancer and promoter elements.
The US region contains the polyadenylation signals. The R (repeat)
region separates the U3 and US regions and transcribed sequences of
the R region appear at both the 5' and 3' ends of the viral
RNA.
[0138] The term "oligonucleotide" as used herein is defined as a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides, preferably at least 5 nucleotides, more preferably
at least about 10-15 nucleotides and more preferably at least about
15 to 30 nucleotides. The exact size will depend on many factors,
which in turn depends on the ultimate function or use of the
oligonucleotide. The oligonucleotide may be generated in any
manner, including chemical synthesis, DNA replication, reverse
transcription, or a combination thereof.
[0139] Because mononucleotides are reacted to make oligonucleotides
in a manner such that the 5' phosphate of one mononucleotide
pentose ring is attached to the 3' oxygen of its neighbor in one
direction via a phosphodiester linkage, an end of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring and
as the "3"end" if its 3' oxygen is not linked to a 5' phosphate of
a subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5' and 3' ends. A first region along a nucleic
acid strand is said to be upstream of another region if the 3' end
of the first region is before the 5' end of the second region when
moving along a strand of nucleic acid in a 5' to 3' direction.
[0140] When two different, non-overlapping oligonucleotides anneal
to different regions of the same linear complementary nucleic acid
sequence, and the 3' end of one oligonucleotide points towards the
5' end of the other, the former may be called the "upstream"
oligonucleotide and the latter the "downstream"
oligonucleotide.
[0141] The term "primer" refers to an oligonucleotide which is
capable of acting as a point of initiation of synthesis when placed
under conditions in which primer extension is initiated. An
oligonucleotide "primer" may occur naturally, as in a purified
restriction digest or may be produced synthetically.
[0142] A primer is selected to be "substantially" complementary to
a strand of specific sequence of the template. A primer must be
sufficiently complementary to hybridize with a template strand for
primer elongation to occur. A primer sequence need not reflect the
exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer,
with the remainder of the primer sequence being substantially
complementary to the strand. Non-complementary bases or longer
sequences can be interspersed into the primer, provided that the
primer sequence has sufficient complementarity with the sequence of
the template to hybridize and thereby form a template primer
complex for synthesis of the extension product of the primer.
[0143] "Hybridization" methods involve the annealing of a
complementary sequence to the target nucleic acid (the sequence to
be detected; the detection of this sequence may be by either direct
or indirect means). The ability of two polymers of nucleic acid
containing complementary sequences to find each other and anneal
through base pairing interaction is a well-recognized phenomenon.
The initial observations of the "hybridization" process by Marmur
and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al.,
Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the
refinement of this process into an essential tool of modern
biology.
[0144] With regard to complementarity, it is important for some
diagnostic applications to determine whether the hybridization
represents complete or partial complementarity. For example, where
it is desired to detect simply the presence or absence of pathogen
DNA (such as from a virus, bacterium, fungi, mycoplasma, protozoan)
it is only important that the hybridization method ensures
hybridization when the relevant sequence is present; conditions can
be selected where both partially complementary probes and
completely complementary probes will hybridize. Other diagnostic
applications, however, may require that the hybridization method
distinguish between partial and complete complementarity. It may be
of interest to detect genetic polymorphisms. For example, human
hemoglobin is composed, in part, of four polypeptide chains. Two of
these chains are identical chains of 141 amino acids (alpha chains)
and two of these chains are identical chains of 146 amino acids
(beta chains). The gene encoding the beta chain is known to exhibit
polymorphism. The normal allele encodes a beta chain having
glutamic acid at the sixth position. The mutant allele encodes a
beta chain having valine at the sixth position. This difference in
amino acids has a profound (most profound when the individual is
homozygous for the mutant allele) physiological impact known
clinically as sickle cell anemia. It is well known that the genetic
basis of the amino acid change involves a single base difference
between the normal allele DNA sequence and the mutant allele DNA
sequence.
[0145] The complement of a nucleic acid sequence as used herein
refers to an oligonucleotide which, when aligned with the nucleic
acid sequence such that the 5' end of one sequence is paired with
the 3' end of the other, is in "antiparallel association." Certain
bases not commonly found in natural nucleic acids may be included
in the nucleic acids of the present invention and include, for
example, inosine and 7-deazaguanine. Complementarity need not be
perfect; stable duplexes may contain mismatched base pairs or
unmatched bases. Those skilled in the art of nucleic acid
technology can determine duplex stability empirically considering a
number of variables including, for example, the length of the
oligonucleotide, base composition and sequence of the
oligonucleotide, ionic strength and incidence of mismatched base
pairs.
[0146] Stability of a nucleic acid duplex is measured by the
melting temperature, or "T.sub.m." The T.sub.m of a particular
nucleic acid duplex under specified conditions is the temperature
at which on average half of the base pairs have disassociated.
[0147] The term "label" as used herein refers to any atom or
molecule which can be used to provide a detectable (preferably
quantifiable) signal, and which can be attached to a nucleic acid
or protein. Labels may provide signals detectable by fluorescence,
radioactivity, colorimetry, gravimetry, X-ray diffraction or
absorption, magnetism, enzymatic activity, and the like. A label
may be a charged moeity (positive or negative charge) or
alternatively, may be charge neutral.
[0148] The term "cleavage structure" as used herein, refers to a
structure which is formed by the interaction of a probe
oligonucleotide and a target nucleic acid to form a duplex, said
resulting structure being cleavable by a cleavage means, including
but not limited to an enzyme. The cleavage structure is a substrate
for specific cleavage by said cleavage means in contrast to a
nucleic acid molecule which is a substrate for non-specific
cleavage by agents such as phosphodiesterases which cleave nucleic
acid molecules without regard to secondary structure (i.e., no
formation of a duplexed structure is required).
[0149] The term "cleavage means" as used herein refers to any means
which is capable of cleaving a cleavage structure, including but
not limited to enzymes. The cleavage means may include native DNAPs
having 5' nuclease activity (e.g., Taq DNA polymerase, E. coli DNA
polymerase I) and, more specifically, modified DNAPs having 5'
nuclease but lacking synthetic activity. The ability of 5'
nucleases to cleave naturally occurring structures in nucleic acid
templates (structure-specific cleavage) is useful to detect
internal sequence differences in nucleic acids without prior
knowledge of the specific sequence of the nucleic acid. In this
manner, they are structure-specific enzymes. "Structure-specific
nucleases" or "structure-specific enzymes" are enzymes which
recognize specific secondary structures in a nucleic molecule and
cleave these structures. The cleavage means of the invention cleave
a nucleic acid molecule in response to the formation of cleavage
structures; it is not necessary that the cleavage means cleave the
cleavage structure at any particular location within the cleavage
structure.
[0150] The cleavage means is not restricted to enzymes having
solely 5' nuclease activity. The cleavage means may include
nuclease activity provided from a variety of sources including the
Cleavase.RTM. enzymes, the FEN-1 endonucleases (including RAD2 and
XPG proteins), Taq DNA polymerase and E. coli DNA polymerase I.
[0151] The term "thermostable" when used in reference to an enzyme,
such as a 5' nuclease, indicates that the enzyme is functional or
active (i.e., can perform catalysis) at an elevated temperature,
i.e., at about 55.degree. C. or higher.
[0152] The term "cleavage products" as used herein, refers to
products generated by the reaction of a cleavage means with a
cleavage structure (i.e., the treatment of a cleavage structure
with a cleavage means).
[0153] The term "target nucleic acid" refers to a nucleic acid
molecule which contains a sequence which has at least partial
complementarity with at least a probe oligonucleotide and may also
have at least partial complementarity with an invader
oligonucleotide. The target nucleic acid may comprise single- or
double-stranded DNA or RNA.
[0154] The term "probe oligonucleotide" refers to an
oligonucleotide which interacts with a target nucleic acid to form
a cleavage structure in the presence or absence of an invader
oligonucleotide. When annealed to the target nucleic acid, the
probe oligonucleotide and target form a cleavage structure and
cleavage occurs within the probe oligonucleotide. In the presence
of an invader oligonucleotide upstream of the probe oligonucleotide
along the target nucleic acid will shift the site of cleavage
within the probe oligonucleotide (relative to the site of cleavage
in the absence of the invader).
[0155] The term "non-target cleavage product" refers to a product
of a cleavage reaction which is not derived from the target nucleic
acid. As discussed above, in the methods of the present invention,
cleavage of the cleavage structure occurs within the probe
oligonucleotide. The fragments of the probe oligonucleotide
generated by this target nucleic acid-dependent cleavage are
"non-target cleavage products."
[0156] The term "invader oligonucleotide" refers to an
oligonucleotide which contains sequences at its 3' end which are
substantially the same as sequences located at the 5' end of a
probe oligonucleotide; these regions will compete for hybridization
to the same segment along a complementary target nucleic acid.
[0157] The term "substantially single-stranded" when used in
reference to a nucleic acid substrate means that the substrate
molecule exists primarily as a single strand of nucleic acid in
contrast to a double-stranded substrate which exists as two strands
of nucleic acid which are held together by inter-strand base
pairing interactions.
[0158] The term "sequence variation" as used herein refers to
differences in nucleic acid sequence between two nucleic acids. For
example, a wild-type structural gene and a mutant form of this
wild-type structural gene may vary in sequence by the presence of
single base substitutions and/or deletions or insertions of one or
more nucleotides. These two forms of the structural gene are said
to vary in sequence from one another. A second mutant form of the
structural gene may exist. This second mutant form is said to vary
in sequence from both the wild-type gene and the first mutant form
of the gene.
[0159] The term "liberating" as used herein refers to the release
of a nucleic acid fragment from a larger nucleic acid fragment,
such as an oligonucleotide, by the action of a 5' nuclease such
that the released fragment is no longer covalently attached to the
remainder of the oligonucleotide.
[0160] The term "K.sub.m" as used herein refers to the
Michaelis-Menten constant for an enzyme and is defined as the
concentration of the specific substrate at which a given enzyme
yields one-half its maximum velocity in an enzyme catalyzed
reaction.
[0161] The term "nucleotide analog" as used herein refers to
modified or non-naturally occurring nucleotides such as 7-deaza
purines (i.e., 7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs
include base analogs and comprise modified forms of
deoxyribonucleotides as well as ribonucleotides.
[0162] The term "polymorphic locus" is a locus present in a
population which shows variation between members of the population
(i.e., the most common allele has a frequency of less than 0.95).
In contrast, a "monomorphic locus" is a genetic locus at little or
no variations seen between members of the population (generally
taken to be a locus at which the most common allele exceeds a
frequency of 0.95 in the gene pool of the population).
[0163] The term "microorganism" as used herein means an organism
too small to be observed with the unaided eye and includes, but is
not limited to bacteria, virus, protozoans, fingi, and
ciliates.
[0164] The term "microbial gene sequences" refers to gene sequences
derived from a microorganism.
[0165] The term "bacteria" refers to any bacterial species
including eubacterial and archaebacterial species.
[0166] The term "virus" refers to obligate, ultramicroscopic,
intracellular parasites incapable of autonomous replication (i.e.,
replication requires the use of the host cell's machinery).
[0167] The term "multi-drug resistant" or multiple-drug resistant"
refers to a microorganism which is resistant to more than one of
the antibiotics or antimicrobial agents used in the treatment of
said microorganism.
[0168] The term "sample" in the present specification and claims is
used in its broadest sense. On the one hand it is meant to include
a specimen or culture (e.g., microbiological cultures). On the
other hand, it is meant to include both biological and
environmental samples.
[0169] Biological samples may be animal, including human, fluid,
solid (e.g., stool) or tissue, as well as liquid and solid food and
feed products and ingredients such as dairy items, vegetables, meat
and meat by-products, and waste. Biological samples may be obtained
from all of the various families of domestic animals, as well as
feral or wild animals, including, but not limited to, such animals
as ungulates, bear, fish, lagamorphs, rodents, etc.
[0170] Environmental samples include environmental material such as
surface matter, soil, water and industrial samples, as well as
samples obtained from food and dairy processing instruments,
apparatus, equipment, utensils, disposable and non-disposable
items. These examples are not to be construed as limiting the
sample types applicable to the present invention.
[0171] The term "source of target nucleic acid" refers to any
sample which contains nucleic acids (RNA or DNA). Particularly
preferred sources of target nucleic acids are biological samples
including, but not limited to blood, saliva, cerebral spinal fluid,
pleural fluid, milk, lymph, sputum and semen.
[0172] An oligonucleotide is said to be present in "excess"
relative to another oligonucleotide (or target nucleic acid
sequence) if that oligonucleotide is present at a higher molar
concentration that the other oligonucleotide (or target nucleic
acid sequence). When an oligonucleotide such as a probe
oligonucleotide is present in a cleavage reaction in excess
relative to the concentration of the complementary target nucleic
acid sequence, the reaction may be used to indicate the amount of
the target nucleic acid present. Typically, when present in excess,
the probe oligonucleotide will be present at least a 100-fold molar
excess; typically at least 1 pmole of each probe oligonucleotide
would be used when the target nucleic acid sequence was present at
about 10 fmoles or less.
[0173] A sample "suspected of containing" a first and a second
target nucleic acid may contain either, both or neither target
nucleic acid molecule.
[0174] The term "charge-balanced" oligonucleotide refers to an
olignucleotide (the input oligonucleotide in a reaction) which has
been modified such that the modified oligonucleotide bears a
charge, such that when the modified oligonucleotide is either
cleaved (i.e., shortened) or elongated, a resulting product bears a
charge different from the input oligonucleotide (the
"charge-unbalanced" oligonucleotide) thereby permitting separation
of the input and reacted oligonucleotides on the basis of charge.
The term "charge-balanced" does not imply that the modified or
balanced oligonucleotide has a net neutral charge (although this
can be the case). Charge-balancing refers to the design and
modification of an oligonucleotide such that a specific reaction
product generated from this input oligonucleotide can be separated
on the basis of charge from the input oligonuceotide.
[0175] For example, in an invader-directed cleavage assay in which
the probe oligonucleotide bears the sequence:
5'-TTCTTTTCACCAGCGAGACGGG-3' (i.e., SEQ ID NO:61 without the
modified bases) and cleavage of the probe occurs between the second
and third residues, one possible charge-balanced version of this
oligonuceotide would be: 5'-Cy3-AminoT-Amino-TCTTTTCACCAGCGAGAC
GGG-3'. This modified oligonucleotide bears a net negative charge.
After cleavage, the following oligonucleotides are generated:
5'-Cy3-AminoT-Amino-T-3' and 5'-CTTTTCACCAGCGAGACGGG-3' (residues
3-22 of SEQ ID NO:61). 5'-Cy3-AminoT-Amino-T-3' bears a detectable
moeity (the positively-charged Cy3 dye) and two amino-modified
bases. The amino-modified bases and the Cy3 dye contribute positive
charges in excess of the negative charges contributed by the
phosphate groups and thus the 5'-Cy3-AminoT-Amino-T-3'
oligonucleotide has a net positive charge. The other, longer
cleavage fragment, like the input probe, bears a net negative
charge. Because the 5'-Cy3-AminoT-Amino-T-3' fragment is separable
on the basis of charge from the input probe (the charge-balanced
oligonucleotide), it is referred to as a charge-unbalanced
oligonucleotide. The longer cleavage product cannot be separated on
the basis of charge from the input oligonucleotide as both
oligonucleotides bear a net negative charge; thus, the longer
cleavage product is not a charge-unbalanced oligonucleotide.
[0176] The term "net neutral charge" when used in reference to an
oligonucletide, including modified oligonucleotides, indicates that
the sum of the charges present (i.e, R--NH.sup.3+ groups on
thymidines, the N3 nitrogen of cytosine, presence or absence or
phosphate groups, etc.) under the desired reaction conditions is
essentially zero. An oligonucletide having a net neutral charge
would not migrate in an electrical field.
[0177] The term "net positive charge" when used in reference to an
oligonucletide, including modified oligonucleotides, indicates that
the sum of the charges present (i.e, R--NH.sup.3+ groups on
thymidines, the N3 nitrogen of cytosine, presence or absence or
phosphate groups, etc.) under the desired reaction conditions is +1
or greater. An oligonucletide having a net positive charge would
migrate toward the negative electrode in an electrical field.
[0178] The term "net negative charge" when used in reference to an
oligonucletide, including modified oligonucleotides, indicates that
the sum of the charges present (i.e, R--NH.sup.3+ groups on
thymidines, the N3 nitrogen of cytosine, presence or absence or
phosphate groups, etc.) under the desired reaction conditions is -1
or lower. An oligonucletide having a net negative charge would
migrate toward the positive electrode in an electrical field.
[0179] The term "polymerization means" refers to any agent capable
of facilitating the addition of nucleoside triphosphates to an
oligonucleotide. Preferred polymerization means comprise DNA
polymerases.
[0180] The term "ligation means" refers to any agent capable of
facilitatig the ligation (i.e., theformation of a phosphodiester
bond between a 3'-OH and a 5'-P located at the termini of two
strands of nuceic acid). Preferred ligation means comprise DNA
ligases and RNA ligases.
[0181] The term "reactant" is used herein in its broadest sense.
The reactant can comprise an enzymatic reactant, a chemical
reactant or ultraviolet light (ultraviolet light, particulary short
wavelength ultraviolet light is known to break oligonucleotide
chains). Any agent capable of reacting with an oligonucleotide to
either shorten (i.e., cleave) or elongate the oligonucleotide is
encompsased within the term "reactant."
[0182] The term "adduct" is used herein in its broadest sense to
indicate any compound or element which can be added to an
oligonucleotide. An adduct may be charged (postively or negatively)
or may be charge neutral. An adduct may be added to the
oligonucleotide via covalent or non-covalent linkages. Examples of
adducts, include but are not limited to indodicarbocyanine dye
amidites, amino-substituted nucleotides, ethidium bromide, ethidium
homodimer, (1,3-propanediamino)propidium,
(diethylenetriamino)propidium, thiazole orange,
(N--N'-tetramethyl-1,3-propanediamino)propyl thiazole orange,
(N--N'-tetramethyl-1,2-ethanediamino)propyl thiazole orange,
thiazole orange-thiazole orange homodimer (TOTO), thiazole
orande-thiazole blue heterodimer (TOTAB), thiazole orange-ethidium
heterodimer 1 (TOED1), thiazole orange-ethidium heterodimer 2
(TOED2) and florescien-ethidium heterodimer (FED), psoralens,
biotin, streptavidin, avidin, etc.
[0183] Where a first oligonucleotide is complementary to a region
of a target nucleic acid and a second oligonucleotide has
complementary to the same region (or a portion of this region) a
"region of overlap" exists along the target nucleic acid. The
degree of overlap will vary depending upon the nature of the
complementarity (see, e.g., region "X" in FIGS. 29 and 67 and the
accompanying discussions).
[0184] As used herein, the term "purified" or "to purify" refers to
the removal of contaminants from a sample. For example, recombinant
Cleavase.RTM. nucleases are expressed in bacterial host cells and
the nucleases are purified by the removal of host cell proteins;
the percent of these recombinant nucleases is thereby increased in
the sample.
[0185] The term "recombinant DNA molecule" as used herein refers to
a DNA molecule which is comprised of segments of DNA joined
together by means of molecular biological techniques.
[0186] The term "recombinant protein" or "recombinant polypeptide"
as used herein refers to a protein molecule which is expressed from
a recombinant DNA molecule.
[0187] As used herein the term "portion" when in reference to a
protein (as in "a portion of a given protein") refers to fragments
of that protein. The fragments may range in size from four amino
acid residues to the entire amino acid sequence minus one ammo
acid.
[0188] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin
which may be single- or double-stranded, and represent the sense or
antisense strand. Similarly, "amino acid sequence" as used herein
refers to peptide or protein sequence.
[0189] "Peptide nucleic acid" ("PNA") as used herein refers to a
molecule which comprises an oligomer to which an amino acid
residue, such as lysine, and an amino group have been added. These
small molecules, also designated anti-gene agents, stop transcript
elongation by binding to their complementary strand of nucleic acid
[Nielsen P E et al. (1993) Anticancer Drug Des. 8:53-63].
[0190] As used herein, the term "substantially purified" refers to
molecules, either nucleic or amino acid sequences, that are removed
from their natural environment, isolated or separated, and are at
least 60% free, preferably 75% free, and most preferably 90% free
from other components with which they are naturally associated. An
"isolated polynucleotide" or "isolated oligonucletide" is therefore
a substantially purified polynucleotide.
DESCRIPTION OF THE INVENTION
[0191] The present invention relates to methods and compositions
for treating nucleic acid, and in particular, methods and
compositions for detection and characterization of nucleic acid
sequences and sequence changes.
[0192] The present invention relates to means for cleaving a
nucleic acid cleavage structure in a site-specific manner. In
particular, the present invention relates to a cleaving enzyme
having 5' nuclease activity without interfering nucleic acid
synthetic ability.
[0193] This invention provides 5' nucleases derived from
thermostable DNA polymerases which exhibit altered DNA synthetic
activity from that of native thermostable DNA polymerases. The 5'
nuclease activity of the polymerase is retained while the synthetic
activity is reduced or absent. Such 5' nucleases are capable of
catalyzing the structure-specific cleavage of nucleic acids in the
absence of interfering synthetic activity. The lack of synthetic
activity during a cleavage reaction results in nucleic acid
cleavage products of uniform size.
[0194] The novel properties of the nucleases of the invention form
the basis of a method of detecting specific nucleic acid sequences.
This method relies upon the amplification of the detection molecule
rather than upon the amplification of the target sequence itself as
do existing methods of detecting specific target sequences.
[0195] DNA polymerases (DNAPs), such as those isolated from E. coli
or from thermophilic bacteria of the genus Thermus, are enzymes
that synthesize new DNA strands. Several of the known DNAPs contain
associated nuclease activities in addition to the synthetic
activity of the enzyme.
[0196] Some DNAPs are known to remove nucleotides from the 5' and
3' ends of DNA chains [Komberg, DNA Replication, W.H. Freeman and
Co., San Francisco, pp. 127-139 (1980)]. These nuclease activities
are usually referred to as 5' exonuclease and 3' exonuclease
activities, respectively. For example, the 5' exonuclease activity
located in the N-terminal domain of several DNAPs participates in
the removal of RNA primers during lagging strand synthesis during
DNA replication and the removal of damaged nucleotides during
repair. Some DNAPs, such as the E. coli DNA polymerase (DNAPEc1),
also have a 3' exonuclease activity responsible for proof-reading
during DNA synthesis (Kornberg, supra).
[0197] A DNAP isolated from Thermus aquaticus, termed Taq DNA
polymerase (DNAPTaq), has a 5' exonuclease activity, but lacks a
functional 3' exonucleolytic domain [Tindall and Kunkell, Biochem.
27:6008 (1988)]. Derivatives of DNAPEc1 and DNAPTaq, respectively
called the Klenow and Stoffel fragments, lack 5' exonuclease
domains as a result of enzymatic or genetic manipulations [Brutlag
et al., Biochem. Biophys. Res. Commun. 37:982 (1969); Erlich et
al., Science 252:1643 (1991); Setlow and Kornberg, J. Biol. Chem.
247:232 (1972)].
[0198] The 5' exonuclease activity of DNAPTaq was reported to
require concurrent synthesis [Gelfand, PCR Technology--Principles
and Applications for DNA Amplification (H. A. Erlich, Ed.),
Stockton Press, New York, p. 19 (1989)]. Although mononucleotides
predominate among the digestion products of the 5' exonucleases of
DNAPTaq and DNAPEc1, short oligonucleotides (<12 nucleotides)
can also be observed implying that these so-called 5' exonucleases
can function endonucleolytically [Setlow, supra; Holland et al.,
Proc. Natl. Acad. Sci. USA 88:7276 (1991)].
[0199] In WO 92/06200, Gelfand et al. show that the preferred
substrate of the 5' exonuclease activity of the thermostable DNA
polymerases is displaced single-stranded DNA. Hydrolysis of the
phosphodiester bond occurs between the displaced single-stranded
DNA and the double-helical DNA with the preferred exonuclease
cleavage site being a phosphodiester bond in the double helical
region. Thus, the 5' exonuclease activity usually associated with
DNAPs is a structure-dependent single-stranded endonuclease and is
more properly referred to as a 5' nuclease. Exonucleases are
enzymes which cleave nucleotide molecules from the ends of the
nucleic acid molecule. Endonucleases, on the other hand, are
enzymes which cleave the nucleic acid molecule at internal rather
than terminal sites. The nuclease activity associated with some
thermostable DNA polymerases cleaves endonucleolytically but this
cleavage requires contact with the 5' end of the molecule being
cleaved. Therefore, these nucleases are referred to as 5'
nucleases.
[0200] When a 5' nuclease activity is associated with a eubacterial
Type A DNA polymerase, it is found in the one-third N-terminal
region of the protein as an independent functional domain. The
C-terminal two-thirds of the molecule constitute the polymerization
domain which is responsible for the synthesis of DNA. Some Type A
DNA polymerases also have a 3' exonuclease activity associated with
the two-third C-terminal region of the molecule.
[0201] The 5' exonuclease activity and the polymerization activity
of DNAPs have been separated by proteolytic cleavage or genetic
manipulation of the polymerase molecule. To date thermostable DNAPs
have been modified to remove or reduce the amount of 5' nuclease
activity while leaving the polymerase activity intact.
[0202] The Klenow or large proteolytic cleavage fragment of DNAPEc1
contains the polymerase and 3' exonuclease activity but lacks the
5' nuclease activity. The Stoffel fragment of DNAPTaq (DNAPStf)
lacks the 5' nuclease activity due to a genetic manipulation which
deleted the N-terminal 289 amino acids of the polymerase molecule
[Erlich et al., Science 252:1643 (1991)]. WO 92/06200 describes a
thermostable DNAP with an altered level of 5' to 3' exonuclease.
U.S. Pat. No. 5,108,892 describes a Thermus aquaticus DNAP without
a 5' to 3' exonuclease. However, the art of molecular biology lacks
a thermostable DNA polymerase with a lessened amount of synthetic
activity.
[0203] The present invention provides 5' nucleases derived from
thermostable Type A DNA polymerases that retain 5' nuclease
activity but have reduced or absent synthetic activity. The ability
to uncouple the synthetic activity of the enzyme from the 5'
nuclease activity proves that the 5' nuclease activity does not
require concurrent DNA synthesis as was previously reported
(Gelfand, PCR Technology, supra).
[0204] The description of the invention is divided into: I.
Detection of Specific Nucleic Acid Sequences Using 5' Nucleases;
II. Generation of 5' Nucleases Derived From Thermostable DNA
Polymerases; III. Detection of Specific Nucleic Acid Sequences
Using 5' Nucleases in an Invader-Directed Cleavage Assay; IV. A
Comparison Of Invasive Cleavage And Primer-Directed Cleavage; V.
Fractionation Of Specific Nucleic Acids By Selective Charge
Reversal; VI. Invader.TM.-Directed Cleavage Using Miniprobes And
Mid-Range Probes; VII. Signal Enhancement By Tailing Of Reaction
Products In The Invader.TM.-Directed Cleavage Assay; VIII. Improved
Enzymes For Use In Invader.TM.-Directed Cleavage Reactions
I. Detection of Specific Nucleic Acid Sequences Using 5'
Nucleases
[0205] The 5' nucleases of the invention form the basis of a novel
detection assay for the identification of specific nucleic acid
sequences. This detection system identifies the presence of
specific nucleic acid sequences by requiring the annealing of two
oligonucleotide probes to two portions of the target sequence. As
used herein, the term "target sequence" or "target nucleic acid
sequence" refers to a specific nucleic acid sequence within a
polynucleotide sequence, such as genomic DNA or RNA, which is to be
either detected or cleaved or both.
[0206] FIG. 1A provides a schematic of one embodiment of the
detection method of the present invention. The target sequence is
recognized by two distinct oligonucleotides in the triggering or
trigger reaction. It is preferred that one of these
oligonucleotides is provided on a solid support. The other can be
provided free. In FIG. 1A the free oligo is indicated as a "primer"
and the other oligo is shown attached to a bead designated as type
1. The target nucleic acid aligns the two oligonucleotides for
specific cleavage of the 5' arm (of the oligo on bead 1) by the
DNAPs of the present invention (not shown in FIG. 1A).
[0207] The site of cleavage (indicated by a large solid arrowhead)
is controlled by the distance between the 3' end of the "primer"
and the downstream fork of the oligo on bead 1. The latter is
designed with an uncleavable region (indicated by the striping). In
this manner neither oligonucleotide is subject to cleavage when
misaligned or when unattached to target nucleic acid.
[0208] Successful cleavage releases a single copy of what is
referred to as the alpha signal oligo. This oligo may contain a
detectable moiety (e.g., fluorescein). On the other hand, it may be
unlabelled.
[0209] In one embodiment of the detection method, two more
oligonucleotides are provided on solid supports. The
oligonucleotide shown in FIG. 1A on bead 2 has a region that is
complementary to the alpha signal oligo (indicated as alpha prime)
allowing for hybridization. This structure can be cleaved by the
DNAPs of the present invention to release the beta signal oligo.
The beta signal oligo can then hybridize to type 3 beads having an
oligo with a complementary region (indicated as beta prime). Again,
this structure can be cleaved by the DNAPs of the present invention
to release a new alpha oligo.
[0210] At this point, the amplification has been linear. To
increase the power of the method, it is desired that the alpha
signal oligo hybridized to bead type 2 be liberated after release
of the beta oligo so that it may go on to hybridize with other
oligos on type 2 beads. Similarly, after release of an alpha oligo
from type 3 beads, it is desired that the beta oligo be
liberated.
[0211] The liberation of "captured" signal oligos can be achieved
in a number of ways. First, it has been found that the DNAPs of the
present invention have a true 5' exonuclease capable of "nibbling"
the 5' end of the alpha (and beta) prime oligo (discussed below in
more detail). Thus, under appropriate conditions, the hybridization
is destabilized by nibbling of the DNAP. Second, the alpha-alpha
prime (as well as the beta-beta prime) complex can be destabilized
by heat (e.g., thermal cycling).
[0212] With the liberation of signal oligos by such techniques,
each cleavage results in a doubling of the number of signal oligos.
In this manner, detectable signal can quickly be achieved.
[0213] FIG. 1B provides a schematic of a second embodiment of the
detection method of the present invention. Again, the target
sequence is recognized by two distinct oligonucleotides in the
triggering or trigger reaction and the target nucleic acid aligns
the two oligonucleotides for specific cleavage of the 5' arm by the
DNAPs of the present invention (not shown in FIG. 1B). The first
oligo is completely complementary to a portion of the target
sequence. The second oligonucleotide is partially complementary to
the target sequence; the 3' end of the second oligonucleotide is
fully complementary to the target sequence while the 5' end is
non-complementary and forms a single-stranded arm. The
non-complementary end of the second oligonucleotide may be a
generic sequence which can be used with a set of standard hairpin
structures (described below). The detection of different target
sequences would require unique portions of two oligonucleotides:
the entire first oligonucleotide and the 3' end of the second
oligonucleotide. The 5' arm of the second oligonucleotide can be
invariant or generic in sequence.
[0214] The annealing of the first and second oligonucleotides near
one another along the target sequence forms a forked cleavage
structure which is a substrate for the 5' nuclease of DNA
polymerases. The approximate location of the cleavage site is again
indicated by the large solid arrowhead in FIG. 1B.
[0215] The 5' nucleases of the invention are capable of cleaving
this structure but are not capable of polymerizing the extension of
the 3' end of the first oligonucleotide. The lack of polymerization
activity is advantageous as extension of the first oligonucleotide
results in displacement of the annealed region of the second
oligonucleotide and results in moving the site of cleavage along
the second oligonucleotide. If polymerization is allowed to occur
to any significant amount, multiple lengths of cleavage product
will be generated. A single cleavage product of uniform length is
desirable as this cleavage product initiates the detection
reaction.
[0216] The trigger reaction may be run under conditions that allow
for thermocycling. Thermocycling of the reaction allows for a
logarithmic increase in the amount of the trigger oligonucleotide
released in the reaction.
[0217] The second part of the detection method allows the annealing
of the fragment of the second oligonucleotide liberated by the
cleavage of the first cleavage structure formed in the triggering
reaction (called the third or trigger oligonucleotide) to a first
hairpin structure. This first hairpin structure has a
single-stranded 5' arm and a single-stranded 3' arm. The third
oligonucleotide triggers the cleavage of this first hairpin
structure by annealing to the 3' arm of the hairpin thereby forming
a substrate for cleavage by the 5' nuclease of the present
invention. The cleavage of this first hairpin structure generates
two reaction products: 1) the cleaved 5' arm of the hairpin called
the fourth oligonucleotide, and 2) the cleaved hairpin structure
which now lacks the 5' arm and is smaller in size than the
uncleaved hairpin. This cleaved first hairpin may be used as a
detection molecule to indicate that cleavage directed by the
trigger or third oligonucleotide occurred. Thus, this indicates
that the first two oligonucleotides found and annealed to the
target sequence thereby indicating the presence of the target
sequence in the sample.
[0218] The detection products are amplified by having the fourth
oligonucleotide anneal to a second hairpin structure. This hairpin
structure has a 5' single-stranded arm and a 3' single-stranded
arm. The fourth oligonucleotide generated by cleavage of the first
hairpin structure anneals to the 3' arm of the second hairpin
structure thereby creating a third cleavage structure recognized by
the 5' nuclease. The cleavage of this second hairpin structure also
generates two reaction products: 1) the cleaved 5' arm of the
hairpin called the fifth oligonucleotide which is similar or
identical in sequence to the third nucleotide, and 2) the cleaved
second hairpin structure which now lacks the 5' arm and is smaller
in size than the uncleaved hairpin. This cleaved second hairpin may
be as a detection molecule and amplifies the signal generated by
the cleavage of the first hairpin structure. Simultaneously with
the annealing of the forth oligonucleotide, the third
oligonucleotide is dissociated from the cleaved first hairpin
molecule so that it is free to anneal to a new copy of the first
hairpin structure. The disassociation of the oligonucleotides from
the hairpin structures may be accomplished by heating or other
means suitable to disrupt base-pairing interactions.
[0219] Further amplification of the detection signal is achieved by
annealing the fifth oligonucleotide (similar or identical in
sequence to the third oligonucleotide) to another molecule of the
first hairpin structure. Cleavage is then performed and the
oligonucleotide that is liberated then is annealed to another
molecule of the second hairpin structure. Successive rounds of
annealing and cleavage of the first and second hairpin structures,
provided in excess, are performed to generate a sufficient amount
of cleaved hairpin products to be detected. The temperature of the
detection reaction is cycled just below and just above the
annealing temperature for the oligonucleotides used to direct
cleavage of the hairpin structures, generally about 55.degree. C.
to 70.degree. C. The number of cleavages will double in each cycle
until the amount of hairpin structures remaining is below the
K.sub.m for the hairpin structures. This point is reached when the
hairpin structures are substantially used up. When the detection
reaction is to be used in a quantitative manner, the cycling
reactions are stopped before the accumulation of the cleaved
hairpin detection products reach a plateau.
[0220] Detection of the cleaved hairpin structures may be achieved
in several ways. In one embodiment detection is achieved by
separation on agarose or polyacrylamide gels followed by staining
with ethidium bromide. In another embodiment, detection is achieved
by separation of the cleaved and uncleaved hairpin structures on a
gel followed by autoradiography when the hairpin structures are
first labelled with a radioactive probe and separation on
chromatography columns using HPLC or FPLC followed by detection of
the differently sized fragments by absorption at OD.sub.260. Other
means of detection include detection of changes in fluorescence
polarization when the single-stranded 5' arm is released by
cleavage, the increase in fluorescence of an intercalating
fluorescent indicator as the amount of primers annealed to 3' arms
of the hairpin structures increases. The formation of increasing
amounts of duplex DNA (between the primer and the 3' arm of the
hairpin) occurs if successive rounds of cleavage occur.
[0221] The hairpin structures may be attached to a solid support,
such as an agarose, styrene or magnetic bead, via the 3' end of the
hairpin. A spacer molecule may be placed between the 3' end of the
hairpin and the bead, if so desired. The advantage of attaching the
hairpin structures to a solid support is that this prevents the
hybridization of the two hairpin structures to one another over
regions which are complementary. If the hairpin structures anneal
to one another, this would reduce the amount of hairpins available
for hybridization to the primers released during the cleavage
reactions. If the hairpin structures are attached to a solid
support, then additional methods of detection of the products of
the cleavage reaction may be employed. These methods include, but
are not limited to, the measurement of the released single-stranded
5' arm when the 5' arm contains a label at the 5' terminus. This
label may be radioactive, fluorescent, biotinylated, etc. If the
hairpin structure is not cleaved, the 5' label will remain attached
to the solid support. If cleavage occurs, the 5' label will be
released from the solid support.
[0222] The 3' end of the hairpin molecule may be blocked through
the use of dideoxynucleotides. A 3' terminus containing a
dideoxynucleotide is unavailable to participate in reactions with
certain DNA modifying enzymes, such as terminal transferase.
Cleavage of the hairpin having a 3' terminal dideoxynucleotide
generates a new, unblocked 3' terminus at the site of cleavage.
This new 3' end has a free hydroxyl group which can interact with
terminal transferase thus providing another means of detecting the
cleavage products.
[0223] The hairpin structures are designed so that their
self-complementary regions are very short (generally in the range
of 3-8 base pairs). Thus, the hairpin structures are not stable at
the high temperatures at which this reaction is performed
(generally in the range of 50-75.degree. C.) unless the hairpin is
stabilized by the presence of the annealed oligonucleotide on the
3' arm of the hairpin. This instability prevents the polymerase
from cleaving the hairpin structure in the absence of an associated
primer thereby preventing false positive results due to
non-oligonucleotide directed cleavage.
[0224] As discussed above, the use of the 5' nucleases of the
invention which have reduced polymerization activity is
advantageous in this method of detecting specific nucleic acid
sequences. Significant amounts of polymerization during the
cleavage reaction would cause shifting of the site of cleavage in
unpredictable ways resulting in the production of a series of
cleaved hairpin structures of various sizes rather than a single
easily quantifiable product. Additionally, the primers used in one
round of cleavage could, if elongated, become unusable for the next
cycle, by either forming an incorrect structure or by being too
long to melt off under moderate temperature cycling conditions. In
a pristine system (i.e., lacking the presence of dNTPs), one could
use the unmodified polymerase, but the presence of nucleotides
(dNTPs) can decrease the per cycle efficiency enough to give a
false negative result. When a crude extract (genomic DNA
preparations, crude cell lysates, etc.) is employed or where a
sample of DNA from a PCR reaction, or any other sample that might
be contaminated with dNTPs, the 5' nucleases of the present
invention that were derived from thermostable polymerases are
particularly useful.
H. Generation of 5' Nucleases From Thermostable DNA Polymerases
[0225] The genes encoding Type A DNA polymerases share about 85%
homology to each other on the DNA sequence level. Preferred
examples of thermostable polymerases include those isolated from
Thermus aquaticus, Thermus flavus, and Thermus thermophilus.
However, other thermostable Type A polymerases which have 5'
nuclease activity are also suitable. FIGS. 2 and 3 compare the
nucleotide and amino acid sequences of the three above mentioned
polymerases. In FIGS. 2 and 3, the consensus or majority sequence
derived from a comparison of the nucleotide (FIG. 2) or amino acid
(FIG. 3) sequence of the three thermostable DNA polymerases is
shown on the top line. A dot appears in the sequences of each of
these three polymerases whenever an amino acid residue in a given
sequence is identical to that contained in the consensus amino acid
sequence. Dashes are used to introduce gaps in order to maximize
alignment between the displayed sequences. When no consensus
nucleotide or amino acid is present at a given position, an "X" is
placed in the consensus sequence. SEQ ID NOS:1-3 display the
nucleotide sequences and SEQ ID NOS:4-6 display the amino acid
sequences of the three wild-type polymerases. SEQ ID NO:1
corresponds to the nucleic acid sequence of the wild type Thermus
aquaticus DNA polymerase gene isolated from the YT-1 strain [Lawyer
et al., J. Biol. Chem. 264:6427 (1989)]. SEQ ID NO:2 corresponds to
the nucleic acid sequence of the wild type Thermus flavus DNA
polymerase gene [Akhmetzjanov and Vakhitov, Nucl. Acids Res.
20:5839 (1992)]. SEQ ID NO:3 corresponds to the nucleic acid
sequence of the wild type Thermus thermophilus DNA polymerase gene
[Gelfand et al., WO 91/09950 (1991)]. SEQ ID NOS:7-8 depict the
consensus nucleotide and amino acid sequences, respectively for the
above three DNAPs (also shown on the top row in FIGS. 2 and 3).
[0226] The 5' nucleases of the invention derived from thermostable
polymerases have reduced synthetic ability, but retain
substantially the same 5' exonuclease activity as the native DNA
polymerase. The term "substantially the same 5"nuclease activity"
as used herein means that the 5' nuclease activity of the modified
enzyme retains the ability to function as a structure-dependent
single-stranded endonuclease but not necessarily at the same rate
of cleavage as compared to the unmodified enzyme. Type A DNA
polymerases may also be modified so as to produce an enzyme which
has increases 5' nuclease activity while having a reduced level of
synthetic activity. Modified enzymes having reduced synthetic
activity and increased 5' nuclease activity are also envisioned by
the present invention.
[0227] By the term "reduced synthetic activity" as used herein it
is meant that the modified enzyme has less than the level of
synthetic activity found in the unmodified or "native" enzyme. The
modified enzyme may have no synthetic activity remaining or may
have that level of synthetic activity that will not interfere with
the use of the modified enzyme in the detection assay described
below. The 5' nucleases of the present invention are advantageous
in situations where the cleavage activity of the polymerase is
desired, but the synthetic ability is not (such as in the detection
assay of the invention).
[0228] As noted above, it is not intended that the invention be
limited by the nature of the alteration necessary to render the
polymerase synthesis deficient. The present invention contemplates
a variety of methods, including but not limited to: 1) proteolysis;
2) recombinant constructs (including mutants); and 3) physical
and/or chemical modification and/or inhibition.
[0229] 1. Proteolysis
[0230] Thermostable DNA polymerases having a reduced level of
synthetic activity are produced by physically cleaving the
unmodified enzyme with proteolytic enzymes to produce fragments of
the enzyme that are deficient in synthetic activity but retain 5'
nuclease activity. Following proteolytic digestion, the resulting
fragments are separated by standard chromatographic techniques and
assayed for the ability to synthesize DNA and to act as a 5'
nuclease. The assays to determine synthetic activity and 5'
nuclease activity are described below.
[0231] 2. Recombinant Constructs
[0232] The examples below describe a preferred method for creating
a construct encoding a 5' nuclease derived from a thermostable DNA
polymerase. As the Type A DNA polymerases are similar in DNA
sequence, the cloning strategies employed for the Thermus aquaticus
and flavus polymerases are applicable to other thermostable Type A
polymerases. In general, a thermostable DNA polymerase is cloned by
isolating genomic DNA using molecular biological methods from a
bacteria containing a thermostable Type A DNA polymerase. This
genomic DNA is exposed to primers which are capable of amplifying
the polymerase gene by PCR.
[0233] This amplified polymerase sequence is then subjected to
standard deletion processes to delete the polymerase portion of the
gene. Suitable deletion processes are described below in the
examples.
[0234] The example below discusses the strategy used to determine
which portions of the DNAPTaq polymerase domain could be removed
without eliminating the 5' nuclease activity. Deletion of amino
acids from the protein can be done either by deletion of the
encoding genetic material, or by introduction of a translational
stop codon by mutation or frame shift. In addition, proteolytic
treatment of the protein molecule can be performed to remove
segments of the protein.
[0235] In the examples below, specific alterations of the Taq gene
were: a deletion between nucleotides 1601 and 2502 (the end of the
coding region), a 4 nucleotide insertion at position 2043, and
deletions between nucleotides 1614 and 1848 and between nucleotides
875 and 1778 (numbering is as in SEQ ID NO:1). These modified
sequences are described below in the examples and at SEQ ID
NOS:9-12.
[0236] Those skilled in the art understand that single base pair
changes can be innocuous in terms of enzyme structure and function.
Similarly, small additions and deletions can be present without
substantially changing the exonuclease or polymerase function of
these enzymes.
[0237] Other deletions are also suitable to create the 5' nucleases
of the present invention. It is preferable that the deletion
decrease the polymerase activity of the 5' nucleases to a level at
which synthetic activity will not interfere with the use of the 5'
nuclease in the detection assay of the invention. Most preferably,
the synthetic ability is absent. Modified polymerases are tested
for the presence of synthetic and 5' nuclease activity as in assays
described below. Thoughtful consideration of these assays allows
for the screening of candidate enzymes whose structure is
heretofore as yet unknown. In other words, construct "X" can be
evaluated according to the protocol described below to determine
whether it is a member of the genus of 5' nucleases of the present
invention as defined functionally, rather than structurally.
[0238] In the example below, the PCR product of the amplified
Thermus aquaticus genomic DNA did not have the identical nucleotide
structure of the native genomic DNA and did not have the same
synthetic ability of the original clone. Base pair changes which
result due to the infidelity of DNAPTaq during PCR amplification of
a polymerase gene are also a method by which the synthetic ability
of a polymerase gene may be inactivated. The examples below and
FIGS. 4A and 5A indicate regions in the native Thermus aquaticus
and flavus DNA polymerases likely to be important for synthetic
ability. There are other base pair changes and substitutions that
will likely also inactivate the polymerase.
[0239] It is not necessary, however, that one start out the process
of producing a 5' nuclease from a DNA polymerase with such a
mutated amplified product. This is the method by which the examples
below were performed to generate the synthesis-deficient DNAPTaq
mutants, but it is understood by those skilled in the art that a
wild-type DNA polymerase sequence may be used as the starting
material for the introduction of deletions, insertion and
substitutions to produce a 5' nuclease. For example, to generate
the synthesis-deficient DNAPTf1 mutant, the primers listed in SEQ
ID NOS:13-14 were used to amplify the wild type DNA polymerase gene
from Thermus flavus strain AT-62. The amplified polymerase gene was
then subjected to restriction enzyme digestion to delete a large
portion of the domain encoding the synthetic activity.
[0240] The present invention contemplates that the nucleic acid
construct of the present invention be capable of expression in a
suitable host. Those in the art know methods for attaching various
promoters and 3' sequences to a gene structure to achieve efficient
expression. The examples below disclose two suitable vectors and
six suitable vector constructs. Of course, there are other
promoter/vector combinations that would be suitable. It is not
necessary that a host organism be used for the expression of the
nucleic acid constructs of the invention. For example, expression
of the protein encoded by a nucleic acid construct may be achieved
through the use of a cell-free in vitro transcription/translation
system. An example of such a cell-free system is the commercially
available TnT.TM. Coupled Reticulocyte Lysate System (Promega
Corporation, Madison, Wis.).
[0241] Once a suitable nucleic acid construct has been made, the 5'
nuclease may be produced from the construct. The examples below and
standard molecular biological teachings enable one to manipulate
the construct by different suitable methods.
[0242] Once the 5' nuclease has been expressed, the polymerase is
tested for both synthetic and nuclease activity as described
below.
[0243] 3. Physical and/or Chemical Modification and/or
Inhibition
[0244] The synthetic activity of a thermostable DNA polymerase may
be reduced by chemical and/or physical means. In one embodiment,
the cleavage reaction catalyzed by the 5' nuclease activity of the
polymerase is run under conditions which preferentially inhibit the
synthetic activity of the polymerase. The level of synthetic
activity need only be reduced to that level of activity which does
not interfere with cleavage reactions requiring no significant
synthetic activity.
[0245] As shown in the examples below, concentrations of Mg.sup.++
greater than 5 mM inhibit the polymerization activity of the native
DNAPTaq. The ability of the 5' nuclease to function under
conditions where synthetic activity is inhibited is tested by
running the assays for synthetic and 5' nuclease activity,
described below, in the presence of a range of Mg.sup.++
concentrations (5 to 10 mM). The effect of a given concentration of
Mg.sup.++ is determined by quantitation of the amount of synthesis
and cleavage in the test reaction as compared to the standard
reaction for each assay.
[0246] The inhibitory effect of other ions, polyamines,
denaturants, such as urea, formamide, dimethylsulfoxide, glycerol
and non-ionic detergents (Triton X-100 and Tween-20), nucleic acid
binding chemicals such as, actinomycin D, ethidium bromide and
psoralens, are tested by their addition to the standard reaction
buffers for the synthesis and 5' nuclease assays. Those compounds
having a preferential inhibitory effect on the synthetic activity
of a thermostable polymerase are then used to create reaction
conditions under which 5' nuclease activity (cleavage) is retained
while synthetic activity is reduced or eliminated.
[0247] Physical means may be used to preferentially inhibit the
synthetic activity of a polymerase. For example, the synthetic
activity of thermostable polymerases is destroyed by exposure of
the polymerase to extreme heat (typically 96 to 100.degree. C.) for
extended periods of time (greater than or equal to 20 minutes).
While these are minor differences with respect to the specific heat
tolerance for each of the enzymes, these are readily determined.
Polymerases are treated with heat for various periods of time and
the effect of the heat treatment upon the synthetic and 5' nuclease
activities is determined.
III. Detection of Specific Nucleic Acid Sequences Using 5'
Nucleases in an Invader-Directed Cleavage Assay
[0248] The present invention provides means for forming a nucleic
acid cleavage structure which is dependent upon the presence of a
target nucleic acid and cleaving the nucleic acid cleavage
structure so as to release distinctive cleavage products. 5'
nuclease activity is used to cleave the target-dependent cleavage
structure and the resulting cleavage products are indicative of the
presence of specific target nucleic acid sequences in the
sample.
[0249] The present invention further provides assays in which the
target nucleic acid is reused or recycled during multiple rounds of
hybridization with oligonucleotide probes and cleavage without the
need to use temperature cycling (i.e., for periodic denaturation of
target nucleic acid strands) or nucleic acid synthesis (i.e., for
the displacement of target nucleic acid strands). Through the
interaction of the cleavage means (e.g., a 5' nuclease) an upstream
oligonucleotide, the cleavage means can be made to cleave a
downstream oligonucleotide at an internal site in such a way that
the resulting fragments of the downstream oligonucleotide
dissociate from the target nucleic acid, thereby making that region
of the target nucleic acid available for hybridization to another,
uncleaved copy of the downstream oligonucleotide.
[0250] As illustrated in FIG. 29, the methods of the present
invention employ at least a pair of oligonucleotides that interact
with a target nucleic acid to form a cleavage structure for a
structure-specific nuclease. More specifically, the cleavage
structure comprises i) a target nucleic acid that may be either
single-stranded or double-stranded (when a double-stranded target
nucleic acid is employed, it may be rendered single stranded, e.g.,
by heating); ii) a first oligonucleotide, termed the "probe," which
defines a first region of the target nucleic acid sequence by being
the complement of that region (regions X and Z of the target as
shown in FIG. 29); iii) a second oligonucleotide, termed the
"invader," the 5' part of which defines a second region of the same
target nucleic acid sequence (regions Y and X in FIG. 29), adjacent
to and downstream of the first target region (regions X and Z), and
the second part of which overlaps into the region defined by the
first oligonucleotide (region X depicts the region of overlap). The
resulting structure is diagrammed in FIG. 29.
[0251] While not limiting the invention or the instant discussion
to any particular mechanism of action, the diagram in FIG. 29
represents the effect on the site of cleavage caused by this type
of arrangement of a pair of oligonucleotides. The design of such a
pair of oligonucleotides is described below in detail. In FIG. 29,
the 3' ends of the nucleic acids (i.e., the target and the
oligonucleotides) are indicated by the use of the arrowheads on the
ends of the lines depicting the strands of the nucleic acids (and
where space permits, these ends are also labelled "3'"). It is
readily appreciated that the two oligonucleotides (the invader and
the probe) are arranged in a parallel orientation relative to one
another, while the target nucleic acid strand is arranged in an
anti-parallel orientation relative to the two oligonucleotides.
Further it is clear that the invader oligonucleotide is located
upstream of the probe oligonucleotide and that with respect to the
target nucleic acid strand, region Z is upstream of region X and
region X is upstream of region Y (that is region Y is downstream of
region X and region X is downstream of region Z). Regions of
complementarity between the opposing strands are indicated by the
short vertical lines. While not intended to indicate the precise
location of the site(s) of cleavage, the area to which the site of
cleavage within the probe oligonucleotide is shifted by the
presence of the invader oligonucleotide is indicated by the solid
vertical arrowhead. An alternative representation of the
target/invader/probe cleavage structure is shown in FIG. 32c.
Neither diagram (i.e., FIG. 29 or FIG. 32c) is intended to
represent the actual mechanism of action or physical arrangement of
the cleavage structure and further it is not intended that the
method of the present invention be limited to any particular
mechanism of action.
[0252] It can be considered that the binding of these
oligonucleotides divides the target nucleic acid into three
distinct regions: one region that has complementarity to only the
probe (shown as "Z"); one region that has complementarity only to
the invader (shown as "Y"); and one region that has complementarity
to both oligonucleotides (shown as "X").
[0253] Design of these oligonucleotides (i.e., the invader and the
probe) is accomplished using practices which are standard in the
art. For example, sequences that have self complementarity, such
that the resulting oligonucleotides would either fold upon
themselves, or hybridize to each other at the expense of binding to
the target nucleic acid, are generally avoided.
[0254] One consideration in choosing a length for these
oligonucleotides is the complexity of the sample containing the
target nucleic acid. For example, the human genome is approximately
3.times.10.sup.9 basepairs in length. Any 10 nucleotide sequence
will appear with a frequency of 1:4.sup.10, or 1:1048,576 in a
random string of nucleotides, which would be approximately 2,861
times in 3 billion basepairs. Clearly an oligonucleotide of this
length would have a poor chance of binding uniquely to a 10
nucleotide region within a target having a sequence the size of the
human genome. If the target sequence were within a 3 kb plasmid,
however, such an oligonucleotide might have a very reasonable
chance of binding uniquely. By this same calculation it can be seen
that an oligonucleotide of 16 nucleotides (i.e., a 16-mer) is the
minimum length of a sequence which is mathematically likely to
appear once in 3.times.10.sup.9 basepairs.
[0255] A second consideration in choosing oligonucleotide length is
the temperature range in which the oligonucleotides will be
expected to function. A 16-mer of average base content (50% G-C
basepairs) will have a calculated T.sub.m (the temperature at which
50% of the sequence is dissociated) of about 41.degree. C.,
depending on, among other things, the concentration of the
oligonucleotide and its target, the salt content of the reaction
and the precise order of the nucleotides. As a practical matter,
longer oligonucleotides are usually chosen to enhance the
specificity of hybridization. Oligonucleotides 20 to 25 nucleotides
in length are often used as they are highly likely to be specific
if used in reactions conducted at temperatures which are near their
T.sub.ms (within about 5.degree. of the T.sub.m). In addition, with
calculated T.sub.ms in the range of 50.degree. to 70.degree. C.,
such oligonucleotides (i.e, 20 to 25-mers) are appropriately used
in reactions catalyzed by thermostable enzymes, which often display
optimal activity near this temperature range.
[0256] The maximum length of the oligonucleotide chosen is also
based on the desired specificity. One must avoid choosing sequences
that are so long that they are either at a high risk of binding
stably to partial complements, or that they cannot easily be
dislodged when desired (e.g., failure to disassociate from the
target once cleavage has occurred).
[0257] The first step of design and selection of the
oligonucleotides for the invader-directed cleavage is in accordance
with these sample general principles. Considered as
sequence-specific probes individually, each oligonucleotide may be
selected according to the guidelines listed above. That is to say,
each oligonucleotide will generally be long enough to be reasonably
expected to hybridize only to the intended target sequence within a
complex sample, usually in the 20 to 40 nucleotide range.
Alternatively, because the invader-directed cleavage assay depends
upon the concerted action of these oligonucleotides, the composite
length of the 2 oligonucleotides which span/bind to the X, Y, Z
regions may be selected to fall within this range, with each of the
individual oligonucleotides being in approximately the 13 to 17
nucleotide range. Such a design might be employed if a
non-thermostable cleavage means were employed in the reaction,
requiring the reactions to be conducted at a lower temperature than
that used when thermostable cleavage means are employed. In some
instances, it may be desirable to have these oligonucleotides bind
multiple times within a target nucleic acid (e.g., which bind to
multiple variants or multiple similar sequences within a target).
It is not intended that the method of the present invention be
limited to any particular size of the probe or invader
oligonucleotide.
[0258] The second step of designing an oligonucleotide pair for
this assay is to choose the degree to which the upstream "invader"
oligonucleotide sequence will overlap into the downstream "probe"
oligonucleotide sequence, and consequently, the sizes into which
the probe will be cleaved. A key feature of this assay is that the
probe oligonucleotide can be made to "turn over," that is to say
cleaved probe can be made to depart to allow the binding and
cleavage of other copies of the probe molecule, without the
requirements of thermal denaturation or displacement by
polymerization. While in one embodiment of this assay probe
turnover may be facilitated by an exonucleolytic digestion by the
cleavage agent, it is central to the present invention that the
turnover does not require this exonucleolytic activity.
[0259] Choosing the Amount of Overlap (Length of the X Region)
[0260] One way of accomplishing such turnover can be envisioned by
considering the diagram in FIG. 29. It can be seen that the T.sub.m
of each oligonucleotide will be a function of the full length of
that oligonucleotide: i.e., the T.sub.m of the invader=Tm(Y+X), and
the T.sub.m of the probe=Tm.sub.(X+Y) for the probe. When the probe
is cleaved the X region is released, leaving the Z section. If the
T.sub.m of Z is less than the reaction temperature, and the
reaction temperature is less than the Tm.sub.(X+Z), then cleavage
of the probe will lead to the departure of Z, thus allowing a new
(X+Z) to hybridize. It can be seen from this example that the X
region must be sufficiently long that the release of X will drop
the T.sub.m of the remaining probe section below the reaction
temperature: a G-C rich X section may be much shorter than an A-T
rich X section and still accomplish this stability shift.
[0261] Designing Oligonucleotides which Interact with the Y and Z
Regions
[0262] If the binding of the invader oligonucleotide to the target
is more stable than the binding of the probe (e.g., if it is long,
or is rich in G-C basepairs in the Y region), then the copy of X
associated with the invader may be favored in the competition for
binding to the X region of the target, and the probe may
consequently hybridize inefficiently, and the assay may give low
signal. Alternatively, if the probe binding is particularly strong
in the Z region, the invader will still cause internal cleavage,
because this is mediated by the enzyme, but portion of the probe
oligonucleotide bound to the Z region may not dissociate at the
reaction temperature, turnover may be poor, and the assay may again
give low signal.
[0263] It is clearly beneficial for the portions of the
oligonucleotide which interact with the Y and Z regions so be
similar in stability, i.e., they must have similar melting
temperatures. This is not to say that these regions must be the
same length. As noted above, in addition to length, the melting
temperature will also be affected by the base content and the
specific sequence of those bases. The specific stability designed
into the invader and probe sequences will depend on the temperature
at which one desires to perform the reaction.
[0264] This discussion is intended to illustrate that (within the
basic guidelines for oligonucleotide specificity discussed above)
it is the balance achieved between the stabilities of the probe and
invader sequences and their X and Y component sequences, rather
than the absolute values of these stabilities, that is the chief
consideration in the selection of the probe and invader
sequences.
[0265] Design of the Reaction Conditions
[0266] Target nucleic acids that may be analyzed using the methods
of the present invention which employ a 5' nuclease as the cleavage
means include many types of both RNA and DNA. Such nucleic acids
may be obtained using standard molecular biological techniques. For
example, nucleic acids (RNA or DNA) may be isolated from a tissue
sample (e.g, a biopsy specimen), tissue culture cells, samples
containing bacteria and/or viruses (including cultures of bacteria
and/or viruses), etc. The target nucleic acid may also be
transcribed in vitro from a DNA template or may be chemically
synthesized or generated in a PCR. Furthermore, nucleic acids may
be isolated from an organism, either as genomic material or as a
plasmid or similar extrachromosomal DNA, or they may be a fragment
of such material generated by treatment with a restriction
endonuclease or other cleavage agents or it may be synthetic.
[0267] Assembly of the target, probe, and invader nucleic acids
into the cleavage reaction of the present invention uses principles
commonly used in the design of oligonucleotide base enzymatic
assays, such as dideoxynucleotide sequencing and polymerase chain
reaction (PCR). As is done in these assays, the oligonucleotides
are provided in sufficient excess that the rate of hybridization to
the target nucleic acid is very rapid. These assays are commonly
performed with 50 fmoles to 2 pmoles of each oligonucleotide per
.mu.l of reaction mixture. In the Examples described herein,
amounts of oligonucleotides ranging from 250 fmoles to 5 pmoles per
.mu.l of reaction volume were used. These values were chosen for
the purpose of ease in demonstration and are not intended to limit
the performance of the present invention to these concentrations.
Other (e.g., lower) oligonucleotide concentrations commonly used in
other molecular biological reactions are also contemplated.
[0268] It is desirable that an invader oligonucleotide be
immediately available to direct the cleavage of each probe
oligonucleotide that hybridizes to a target nucleic acid. For this
reason, in the Examples described herein, the invader
oligonucleotide is provided in excess over the probe
oligonucleotide; often this excess is 10-fold. While this is an
effective ratio, it is not intended that the practice of the
present invention be limited to any particular ratio of
invader-to-probe (a ratio of 2- to 100-fold is contemplated).
[0269] Buffer conditions must be chosen that will be compatible
with both the oligonucleotide/target hybridization and with the
activity of the cleavage agent. The optimal buffer conditions for
nucleic acid modification enzymes, and particularly DNA
modification enzymes, generally included enough mono- and di-valent
salts to allow association of nucleic acid strands by base-pairing.
If the method of the present invention is performed using an
enzymatic cleavage agent other than those specifically described
here, the reactions may generally be performed in any such buffer
reported to be optimal for the nuclease function of the cleavage
agent. In general, to test the utility of any cleavage agent in
this method, test reactions are performed wherein the cleavage
agent of interest is tested in the MOPS/MnCl.sub.2/KCl buffer or
Mg-containing buffers described herein and in whatever buffer has
been reported to be suitable for use with that agent, in a
manufacturer's data sheet, a journal article, or in personal
communication.
[0270] The products of the invader-directed cleavage reaction are
fragments generated by structure-specific cleavage of the input
oligonucleotides. The resulting cleaved and/or uncleaved
oligonucleotides may be analyzed and resolved by a number of
methods including electrophoresis (on a variety of supports
including acrylamide or agarose gels, paper, etc.), chromatography,
fluorescence polarization, mass spectrometry and chip
hybridization. The invention is illustrated using electrophoretic
separation for the analysis of the products of the cleavage
reactions. However, it is noted that the resolution of the cleavage
products is not limited to electrophoresis. Electrophoresis is
chosen to illustrate the method of the invention because
electrophoresis is widely practiced in the art and is easily
accessible to the average practioner.
[0271] The probe and invader oligonucleotides may contain a label
to aid in their detection following the cleavage reaction. The
label may be a radioisotope (e.g., a .sup.32P or .sup.35S-labelled
nucleotide) placed at either the 5' or 3' end of the
oligonucleotide or alternatively, the label may be distributed
throughout the oligonucleotide (i.e., a uniformly labelled
oligonucleotide). The label may be a nonisotopic detectable moiety,
such as a fluorophore, which can be detected directly, or a
reactive group which permits specific recognition by a secondary
agent. For example, biotinylated oligonucleotides may be detected
by probing with a streptavidin molecule which is coupled to an
indicator (e.g., alkaline phosphatase or a fluorophore) or a hapten
such as dioxigenin may be detected using a specific antibody
coupled to a similar indicator.
[0272] Optimization of Reaction Conditions
[0273] The invader-directed cleavage reaction is useful to detect
the presence of specific nucleic acids. In addition to the
considerations listed above for the selection and design of the
invader and probe oligonucleotides, the conditions under which the
reaction is to be performed may be optimized for detection of a
specific target sequence.
[0274] One objective in optimizing the invader-directed cleavage
assay is to allow specific detection of the fewest copies of a
target nucleic acid. To achieve this end, it is desirable that the
combined elements of the reaction interact with the maximum
efficiency, so that the rate of the reaction (e.g., the number of
cleavage events per minute) is maximized. Elements contributing to
the overall efficiency of the reaction include the rate of
hybridization, the rate of cleavage, and the efficiency of the
release of the cleaved probe.
[0275] The rate of cleavage will be a function of the cleavage
means chosen, and may be made optimal according to the
manufacturer's instructions when using commercial preparations of
enzymes or as described in the examples herein. The other elements
(rate of hybridization, efficiency of release) depend upon the
execution of the reaction, and optimization of these elements is
discussed below.
[0276] Three elements of the cleavage reaction that significantly
affect the rate of nucleic acid hybridization are the concentration
of the nucleic acids, the temperature at which the cleavage
reaction is performed and the concentration of salts and/or other
charge-shielding ions in the reaction solution.
[0277] The concentrations at which oligonucleotide probes are used
in assays of this type are well known in the art, and are discussed
above. One example of a common approach to optimizing an
oligonucleotide concentration is to choose a starting amount of
oligonucleotide for pilot tests; 0.01 to 2 .mu.M is a concentration
range used in many oligonucleotide-based assays. When initial
cleavage reactions are performed, the following questions may be
asked of the data: Is the reaction performed in the absence of the
target nucleic acid substantially free of the cleavage product?; Is
the site of cleavage specifically shifted in accordance with the
design of the invader oligonucleotide?; Is the specific cleavage
product easily detected in the presence of the uncleaved probe (or
is the amount of uncut material overwhelming the chosen
visualization method)?
[0278] A negative answer to any of these questions would suggest
that the probe concentration is too high, and that a set of
reactions using serial dilutions of the probe should be performed
until the appropriate amount is identified. Once identified for a
given target nucleic acid in a give sample type (e.g., purified
genomic DNA, body fluid extract, lysed bacterial extract), it
should not need to be re-optimized. The sample type is important
because the complexity of the material present may influence the
probe optimum.
[0279] Conversely, if the chosen initial probe concentration is too
low, the reaction may be slow, due to inefficient hybridization.
Tests with increasing quantities of the probe will identify the
point at which the concentration exceeds the optimum. Since the
hybridization will be facilitated by excess of probe, it is
desirable, but not required, that the reaction be performed using
probe concentrations just below this point.
[0280] The concentration of invader oligonucleotide can be chosen
based on the design considerations discussed above. In a preferred
embodiment, the invader oligonucleotide is in excess of the probe
oligonucleotide. In a particularly preferred embodiment, the
invader is approximately 10-fold more abundant than the probe.
[0281] Temperature is also an important factor in the hybridization
of oligonucleotides. The range of temperature tested will depend in
large part, on the design of the oligonucleotides, as discussed
above. In a preferred embodiment, the reactions are performed at
temperatures slightly below the T.sub.m of the least stable
oligonucleotide in the reaction. Melting temperatures for the
oligonucleotides and for their component regions (X, Y and Z, FIG.
29), can be estimated through the use of computer software or, for
a more rough approximation, by assigning the value of 2.degree. C.
per A-T basepair, and 4.degree. C. per G-C basepair, and taking the
sum across an expanse of nucleic acid. The latter method may be
used for oligonucleotides of approximately 10-30 nucleotides in
length. Because even computer prediction of the T.sub.m of a
nucleic acid is only an approximation, the reaction temperatures
chosen for initial tests should bracket the calculated T.sub.m.
While optimizations are not limited to this, 5.degree. C.
increments are convenient test intervals in these optimization
assays.
[0282] When temperatures are tested, the results can be analyzed
for specificity (the first two of the questions listed above) in
the same way as for the oligonucleotide concentration
determinations. Non-specific cleavage (i.e., cleavage of the probe
at many or all positions along its length) would indicate
non-specific interactions between the probe and the sample
material, and would suggest that a higher temperature should be
employed. Conversely, little or no cleavage would suggest that even
the intended hybridization is being prevented, and would suggest
the use of lower temperatures. By testing several temperatures, it
is possible to identify an approximate temperature optimum, at
which the rate of specific cleavage of the probe is highest. If the
oligonucleotides have been designed as described above, the T.sub.m
of the Z-region of the probe oligonucleotide should be below this
temperature, so that turnover is assured.
[0283] A third determinant of hybridization efficiency is the salt
concentration of the reaction. In large part, the choice of
solution conditions will depend on the requirements of the cleavage
agent, and for reagents obtained commercially, the manufacturer's
instructions are a resource for this information. When developing
an assay utilizing any particular cleavage agent, the
oligonucleotide and temperature optimizations described above
should be performed in the buffer conditions best suited to that
cleavage agent.
[0284] A "no enzyme" control allows the assessment of the stability
of the labeled oligonucleotides under particular reaction
conditions, or in the presence of the sample to be tested (i.e., in
assessing the sample for contaminating nucleases). In this manner,
the substrate and oligonucleotides are placed in a tube containing
all reaction components, except the enzyme and treated the same as
the enzyme-containing reactions. Other controls may also be
included. For example, a reaction with all of the components except
the target nucleic acid will serve to confirm the dependence of the
cleavage on the presence of the target sequence.
[0285] Probing for Multiple Alleles
[0286] The invader-directed cleavage reaction is also useful in the
detection and quantification of individual variants or alleles in a
mixed sample population. By way of example, such a need exists in
the analysis of tumor material for mutations in genes associated
with cancers. Biopsy material from a tumor can have a significant
complement of normal cells, so it is desirable to detect mutations
even when present in fewer than 5% of the copies of the target
nucleic acid in a sample. In this case, it is also desirable to
measure what fraction of the population carries the mutation.
Similar analyses may also be done to examine allelic variation in
other gene systems, and it is not intended that the method of the
present invention by limited to the analysis of tumors.
[0287] As demonstrated below, reactions can be performed under
conditions that prevent the cleavage of probes bearing even a
single-nucleotide difference mismatch within the region of the
target nucleic acid termed "Z" in FIG. 29, but that permit cleavage
of a similar probe that is completely complementary to the target
in this region. Thus, the assay may be used to quantitate
individual variants or alleles within a mixed sample.
[0288] The use of multiple, differently labelled probes in such an
assay is also contemplated. To assess the representation of
different variants or alleles in a sample, one would provide a
mixture of probes such that each allele or variant to be detected
would have a specific probe (i.e., perfectly matched to the Z
region of the target sequence) with a unique label (e.g., no two
variant probes with the same label would be used in a single
reaction). These probes would be characterized in advance to ensure
that under a single set of reaction conditions, they could be made
to give the same rate of signal accumulation when mixed with their
respective target nucleic acids. Assembly of a cleavage reaction
comprising the mixed probe set, a corresponding invader
oligonucleotide, the target nucleic acid sample, and the
appropriate cleavage agent, along with performance of the cleavage
reaction under conditions such that only the matched probes would
cleave, would allow independent quantification of each of the
species present, and would therefore indicate their relative
representation in the target sample.
IV. A Comparision of Invasive Cleavage And Primer-Directed
Cleavage
[0289] As discussed herein, the terms "invasive" or
"invader-directed" cleavage specifically denote the use of a first,
upstream oligonucleotide, as defined below, to cause specific
cleavage at a site within a second, downstream sequence. To effect
such a direction of cleavage to a region within a duplex, it is
required that the first and second oligonucleotides overlap in
sequence. That is to say, a portion of the upstream
oligonucleotide, termed the "invader", has significant homology to
a portion of the downstream "probe" oligonucleotide, so that these
regions would tend to basepair with the same complementary region
of the target nucleic acid to be detected. While not limiting the
present invention to any particular mechanism, the overlapping
regions would be expected to alternate in their occupation of the
shared hybridization site. When the probe oligonucleotide fully
anneals to the target nucleic acid, and thus forces the 3' region
of the invader to remain unpaired, the structure so formed is not a
substrate for the 5' nucleases of the present invention. By
contrast, when the inverse is true, the structure so formed is
substrate for these enzymes, allowing cleavage and release of the
portion of the probe oligonucleotide that is displaced by the
invader oligonucleotide. The shifting of the cleavage site to a
region the probe oligonucleotide that would otherwise be basepaired
to the target sequence is one hallmark of the invasive cleavage
assay (i.e., the invader-directed cleavage assay) of the present
invention.
[0290] It is beneficial at this point to contrast the invasive
cleavage as described above with two other forms of probe cleavage
that may lead to internal cleavage of a probe oligonucleotide, but
which do not comprise invasive cleavage. In the first case, a
hybridized probe may be subject to duplex-dependent 5' to 3'
exonuclease "nibbling," such that the oligonucleotide is shortened
from the 5' end until it cannot remain bound to the target (see,
e.g., Examples 6-8 and FIGS. 26-28). The site at which such
nibbling stops can appear to be discrete, and, depending on the
difference between the melting temperature of the full-length probe
and the temperature of the reaction, this stopping point may be 1
or several nucleotides into the probe oligonucleotide sequence.
Such "nibbling" is often indicated by the presence of a "ladder" of
longer products ascending size up to that of the full length of the
probe, but this is not always the case. While any one of the
products of such a nibbling reaction may be made to match in size
and cleavage site the products of an invasive cleavage reaction,
the creation of these nibbling products would be highly dependent
on the temperature of the reaction and the nature of the cleavage
agent, but would be independent of the action of an upstream
oligonucleotide, and thus could not be construed to involve
invasive cleavage.
[0291] A second cleavage structure that may be considered is one in
which a probe oligonucleotide has several regions of
complementarity with the target nucleic acid, interspersed with one
or more regions or nucleotides of noncomplementarity. These
noncomplementary regions may be thought of as "bubbles" within the
nucleic acid duplex. As temperature is elevated, the regions of
complementarity can be expected to "melt" in the order of their
stability, lowest to highest. When a region of lower stability is
near the end of a segment of duplex, and the next region of
complementarity along the strand has a higher melting temperature,
a temperature can be found that will cause the terminal region of
duplex to melt first, opening the first bubble, and thereby
creating a preferred substrate structure of the cleavage by the 5'
nucleases of the present invention (FIG. 40a). The site of such
cleavage would be expected to be on the 5' arm, within 2
nucleotides of the junction between the single and double-stranded
regions (Lyamichev et al., supra. and U.S. Pat. No. 5,422,253)
[0292] An additional oligonucleotide could be introduced to
basepair along the target nucleic acid would have a similar effect
of opening this bubble for subsequent cleavage of the unpaired 5'
arm (FIG. 40b and FIG. 6). Note in this case, the 3' terminal
nucleotides of the upstream oligonucleotide anneals along the
target nucleic acid sequence in such a manner that the 3' end is
located within the "bubble" region. Depending on the precise
location of the 3' end of this oligonucleotide, the cleavage site
may be along the newly unpaired 5' arm, or at the site expected for
the thermally opened bubble structure as described above. In the
former case the cleavage is not within a duplexed region, and is
thus not invasive cleavage, while in the latter the oligonucleotide
is merely an aide in inducing cleavage at a site that might
otherwise be exposed through the use of temperature alone (i.e., in
the absence of the additional oligonucleotide), and is thus not
considered to be invasive cleavage.
[0293] In summary, any arrangement of oligonucleotides used for the
cleavage-based detection of a target sequence can be analyzed to
determine if the arrangement is an invasive cleavage structure as
contemplated herein. An invasive cleavage structure supports
cleavage of the probe in a region that, in the absence of an
upstream oligonucleotide, would be expected to be basepaired to the
target nucleic acid.
[0294] Example 26 below provides further guidance for the design
and execution of a experiments which allow the determination of
whether a given arrangement of a pair of upstream and downstream
(i.e., the probe) oligonucleotides when annealed along a target
nucleic acid would form an invasive cleavage structure.
V. Fractionation of Specific Nucleic Acids by Selective Charge
Reversal
[0295] Some nucleic acid-based detection assays involve the
elongation and/or shortening of oligonucleotide probes. For
example, as described herein, the primer-directed,
primer-independent, and invader-directed cleavage assays, as well
as the "nibbling" assay all involve the cleavage (i.e., shortening)
of oligonucleotides as a means for detecting the presence of a
target nucleic sequence. Examples of other detection assays which
involve the shortening of an oligonucleotide probe include the
"TaqMan" or nick-translation PCR assay described in U.S. Pat. No.
5,210,015 to Gelfand et al. (the disclosure of which is herein
incorporated by reference), the assays described in U.S. Pat. Nos.
4,775,619 and 5,118,605 to Urdea (the disclosures of which are
herein incorporated by reference), the catalytic hybridization
amplification assay described in U.S. Pat. No. 5,403,711 to Walder
and Walder (the disclosure of which is herein incorporated by
reference), and the cycling probe assay described in U.S. Pat. Nos.
4,876,187 and 5,011,769 to Duck et al. (the disclosures of which
are herein incorporated by reference). Examples of detection assays
which involve the elongation of an oligonucleotide probe (or
primer) include the polymerase chain reaction (PCR) described in
U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al.
(the disclosures of which are herein incorporated by reference) and
the ligase chain reaction (LCR) described in U.S. Pat. Nos.
5,427,930 and 5,494,810 to Birkenmeyer et al. and Barany et al.
(the disclosures of which are herein incorporated by reference).
The above examples are intended to be illustrative of nucleic
acid-based detection assays that involve the elongation and/or
shortening of oligonucleotide probes and do not provide an
exhaustive list.
[0296] Typically, nucleic acid-based detection assays that involve
the elongation and/or shortening of oligonucleotide probes require
post-reaction analysis to detect the products of the reaction. It
is common that, the specific reaction product(s) must be separated
from the other reaction components, including the input or
unreacted oligonucleotide probe. One detection technique involves
the electrophoretic separation of the reacted and unreacted
oligonucleotide probe. When the assay involves the cleavage or
shortening of the probe, the unreacted product will be longer than
the reacted or cleaved product. When the assay involves the
elongation of the probe (or primer), the reaction products will be
greater in length than the input. Gel-based electrophoresis of a
sample containing nucleic acid molecules of different lengths
separates these fragments primarily on the basis of size. This is
due to the fact that in solutions having a neutral or alkaline pH,
nucleic acids having widely different sizes (i.e., molecular
weights) possess very similar charge-to-mass ratios and do not
separate [Andrews, Electrophoresis, 2nd Edition, Oxford University
Press (1986), pp. 153-154]. The gel matrix acts as a molecular
sieve and allows nucleic acids to be separated on the basis of size
and shape (e.g., linear, relaxed circular or covalently closed
supercoiled circles).
[0297] Unmodified nucleic acids have a net negative charge due to
the presence of negatively charged phosphate groups contained
within the sugar-phosphate backbone of the nucleic acid. Typically,
the sample is applied to gel near the negative pole and the nucleic
acid fragments migrate into the gel toward the positive pole with
the smallest fragments moving fastest through the gel.
[0298] The present invention provides a novel means for
fractionating nucleic acid fragments on the basis of charge. This
novel separation technique is related to the observation that
positively charged adducts can affect the electrophoretic behavior
of small oligonucleotides because the charge of the adduct is
significant relative to charge of the whole complex. In addition,
to the use of positively charged adducts (e.g. Cy3 and Cy5 amidite
fluorescent dyes, the positively charged heterodimeric DNA-binding
dyes shown in FIG. 66, etc.), the oligonucleotide may contain amino
acids (particulary useful amino acids are the charged amino acids:
lysine, arginine, asparate, glutamate), modified bases, such as
amino-modified bases, and/or a phosphonate backbone (at all or a
subset of the positions). In addition as discussed further below, a
neutral dye or detection moiety (e.g., biotin, streptavidin, etc.)
may be employed in place of a positively charged adduct in
conjunction with the use of amino-modified bases and/or a complete
or partial phosphonate backbone.
[0299] This observed effect is of particular utility in assays
based on the cleavage of DNA molecules. Using the assays described
herein as an example, when an oligonucleotide is shortened through
the action of a Cleavase@ enzyme or other cleavage agent, the
positive charge can be made to not only significantly reduce the
net negative charge, but to actually override it, effectively
"flipping" the net charge of the labeled entity. This reversal of
charge allows the products of target-specific cleavage to be
partitioned from uncleaved probe by extremely simple means. For
example, the products of cleavage can be made to migrate towards a
negative electrode placed at any point in a reaction vessel, for
focused detection without gel-based electrophoresis; Example 24
provides examples of devices suitable for focused detection without
gel-based electrophoresis. When a slab gel is used, sample wells
can be positioned in the center of the gel, so that the cleaved and
uncleaved probes can be observed to migrate in opposite directions.
Alternatively, a traditional vertical gel can be used, but with the
electrodes reversed relative to usual DNA gels (i.e., the positive
electrode at the top and the negative electrode at the bottom) so
that the cleaved molecules enter the gel, while the uncleaved
disperse into the upper reservoir of electrophoresis buffer.
[0300] An important benefit of this type of readout is the absolute
nature of the partition of products from substrates, i.e., the
separation is virtually 100%. This means that an abundance of
uncleaved probe can be supplied to drive the hybridization step of
the probe-based assay, yet the unconsumed (i.e., unreacted) probe
can, in essence, be subtracted from the result to reduce background
by virtue of the fact that the unreacted probe will not migrate to
the same pole as the specific reaction product.
[0301] Through the use of multiple positively charged adducts,
synthetic molecules can be constructed with sufficient modification
that the normally negatively charged strand is made nearly neutral.
When so constructed, the presence or absence of a single phosphate
group can mean the difference between a net negative or a net
positive charge. This observation has particular utility when one
objective is to discriminate between enzymatically generated
fragments of DNA, which lack a 3 phosphate, and the products of
thermal degradation, which retain a 3 phosphate (and thus two
additional negative charges). Examples 23 and 24 demonstrate the
ability to separate positively charged reaction products from a net
negatively charged substrate oligonucleotide. As discussed in these
examples, oligonucleotides may be transformed from net negative to
net positively charged compounds. In Example 24, the positively
charged dye, Cy3 was incorporated at the 5' end of a 22-mer (SEQ ID
NO:61) which also contained two amino-substituted residues at the
5' end of the oligonucleotide; this oligonucleotide probe carries a
net negative charge. After cleavage, which occurred 2 nucleotides
into the probe, the following labelled oligonucleotide was
released: 5'-Cy3-AminoT-AminoT-3' (as well as the remaining 20
nucleotides of SEQ ID NO:61). This short fragment bears a net
positive charge while the reaminder of the cleaved oligonucleotide
and the unreacted or input oligonucleotide bear net negative
charges.
[0302] The present invention contemplates embodiments wherein the
specific reaction product produced by any cleavage of any
oligonucleotide can be designed to carry a net positive charge
while the unreacted probe is charge neutral or carries a net
negative charge. The present invention also contemplates
embodiments where the released product may be designed to carry a
net negative charge while the input nucleic acid carries a net
positive charge. Depending on the length of the released product to
be detected, positively charged dyes may be incorporated at the one
end of the probe and modified bases may be placed along the
oligonucleotide such that upon cleavage, the released fragment
containing the positively charged dye carries a net positive
charge. Amino-modified bases may be used to balance the charge of
the released fragment in cases where the presence of the positively
charged adduct (e.g., dye) alone is not sufficient to impart a net
positive charge on the released fragment. In addition, the
phosphate backbone may be replaced with a phosphonate backbone at a
level sufficient to impart a net positive charge (this is
particularly useful when the sequence of the oligonucleotide is not
amenable to the use of amino-substituted bases); FIGS. 56 and 57
show the structure of short oligonucleotides containing a
phosphonate group on the second T residue). An oligonucleotide
containing a fully phosphonate-substituted backbone would be charge
neutral (absent the presence of modified charged residues bearing a
charge or the presence of a charged adduct) due to the absence of
the negatively charged phosphate groups. Phosphonate-containing
nucleotides (e.g., methylphosphonate-containing nucleotides are
readily available and can be incorporated at any position of an
oligonucleotide during synthesis using techniques which are well
known in the art.
[0303] In essence, the invention contemplates the use of
charge-based separation to permit the separation of specific
reaction products from the input oligonucleotides in nucleic
acid-based detection assays. The foundation of this novel
separation technique is the design and use of oligonucleotide
probes (typically termed "primers" in the case of PCR) which are
"charge balanced" so that upon either cleavage or elongation of the
probe it becomes "charge unbalanced," and the specific reaction
products may be separated from the input reactants on the basis of
the net charge.
[0304] In the context of assays which involve the elongation of an
oligonucleotide probe (i.e., a primer), such as is the case in PCR,
the input primers are designed to carry a net positive charge.
Elongation of the short oligonucleotide primer during
polymerization will generate PCR products which now carry a net
negative charge. The specific reaction products may then easily be
separated and concentrated away from the input primers using the
charge-based separation technique described herein (the electrodes
will be reversed relative to the description in Example 24 as the
product to be separated and concentrated after a PCR will carry a
negative charge).
VI. Invader.TM.-Directed Cleavage Using Miniprobes And Mid-Range
Probes
[0305] As discussed in section III above, the Invader.TM.-directed
cleavage assay may be performed using inavder and probe
oligonucleotides which have a length of about 13-25 nucleotides
(typically 20-25 nucleotides). It is also contemplated that the
oligonucleotides that span the X, Y and Z regions (see FIG. 29),
the invader and probe oligonucleotides, may themselves be composed
of shorter oligonucleotide sequences that align along a target
strand but that are not covalently linked. This is to say that
there is a nick in the sugar-phosphate backbone of the composite
oligonucleotide, but that there is no disruption in the progression
of base-paired nucleotides in the resulting duplex. When short
strands of nucleic acid align contiguously along a longer strand
the hybridization of each is stabilized by the hybridization of the
neighboring fragments because the basepairs can stack along the
helix as though the backbone was in fact uninterrupted. This
cooperativity of binding can give each segment a stability of
interaction in excess of what would be expected for the segment
hybridizing to the longer nucleic acid alone. One application of
this observation has been to assemble primers for DNA sequencing,
typically about 18 nucleotides long, from sets of three hexamer
oligonucleotides that are designed to hybridize in this way
[Kotler, L. E., et al. (1993) Proc. Natl. Acad. Sci. USA 90:4241].
The resulting doubly-nicked primer can be extended enzymatically in
reactions performed at temperatures that might be expected to
disrupt the hybridization of hexamers, but not of 18-mers.
[0306] The use of composite or split oligonuceotides is applied
with success in the Invader.TM.-directed cleavage assay. The probe
oligonucleotide may be split into two oligonucleotides which anneal
in a contigious and adjacent manner along a target oligonucleotide
as diagrammed in FIG. 68. In this figure, the downstream
oligonucleotide (analogous to the probe of FIG. 29) is assembled
from two smaller pieces: a short segment of 6-10 nts (termed the
"miniprobe"), that is to be cleaved in the course of the detection
reaction, and an oligonucleotide that hybridizes immediately
downstream of the miniprobe (termed the "stacker"), which serves to
stabilize the hybridization of the probe. To form the cleavage
structure, an upstream oligonucleotide (the "Invader.TM." oligo) is
provided to direct the cleavage activity to the desired region of
the miniprobe. Assembly of the probe from non-linked pieces of
nucleic acid (i.e., the miniprobe and the stacker) allows regions
of sequences to be changed without requiring the re-synthesis of
the entire proven sequence, thus improving the cost and flexibility
of the detection system. In addition, the use of unlinked composite
oligonucleotides makes the system more stringent in its requirement
of perfectly matched hybridization to achieve signal generation,
allowing this to be used as a sensitive means of detecting
mutations or changes in the target nucleic acid sequences.
[0307] As illustrated in FIG. 68, in one embodiment, the methods of
the present invention employ at least three oligonucleotides that
interact with a target nucleic acid to form a cleavage structure
for a structure-specific nuclease. More specifically, the cleavage
structure comprises i) a target nucleic acid that may be either
single-stranded or double-stranded (when a double-stranded target
nucleic acid is employed, it may be rendered single-stranded, e.g.,
by heating); ii) a first oligonucleotide, termed the "stacker,"
which defines a first region of the target nucleic acid sequence by
being the complement of that region (region W of the target as
shown in FIG. 67); iii) a second oligonucleotide, termed the
"miniprobe," which defines a second region of the target nucleic
acid sequence by being the complement of that region (regions X and
Z of the target as shown in FIG. 67); iv) a third oligonucleotide,
termed the "invader," the 5' part of which defines a third region
of the same target nucleic acid sequence (regions Y and X in FIG.
67), adjacent to and downstream of the second target region
(regions X and Z), and the second or 3' part of which overlaps into
the region defined by the second oligonucleotide (region X depicts
the region of overlap). The resulting structure is diagrammed in
FIG. 68.
[0308] While not limiting the invention or the instant discussion
to any particular mechanism of action, the diagram in FIG. 68
represents the effect on the site of cleavage caused by this type
of arrangement of three oligonucleotides. The design of these three
oligonucleotides is described below in detail. In FIG. 68, the 3'
ends of the nucleic acids (i.e., the target and the
oligonucleotides) are indicated by the use of the arrowheads on the
ends of the lines depicting the strands of the nucleic acids (and
where space permits, these ends are also labelled "3'"). It is
readily appreciated that the three oligonucleotides (the invader,
the miniprobe and the stacker) are arranged in a parallel
orientation relative to one another, while the target nucleic acid
strand is arranged in an anti-parallel orientation relative to the
three oligonucleotides. Further it is clear that the invader
oligonucleotide is located upstream of the miniprobe
oligonucleotide and that the miniprobe olignuceotide is located
upstream of the stacker oligonucleotide and that with respect to
the target nucleic acid strand, region W is upstream of region Z,
region Z is upstream of upstream of region X and region X is
upstream of region Y (that is region Y is downstream of region X,
region X is downstream of region Z and region Z is downstream of
region W). Regions of complementarity between the opposing strands
are indicated by the short vertical lines. While not intended to
indicate the precise location of the site(s) of cleavage, the area
to which the site of cleavage within the miniprobe oligonucleotide
is shifted by the presence of the invader oligonucleotide is
indicated by the solid vertical arrowhead. FIG. 68 is not intended
to represent the actual mechanism of action or physical arrangement
of the cleavage structure and further it is not intended that the
method of the present invention be limited to any particular
mechanism of action.
[0309] It can be considered that the binding of these
oligonucleotides divides the target nucleic acid into four distinct
regions: one region that has complementarity to only the stacker
(shown as "W"); one region that has complemetarity to only the
miniprobe (shown as "Z"); one region that has complementarity only
to the Invader.TM. oligo (shown as "Y"); and one region that has
complementarity to both the Invader.TM. and miniprobe
oligonucleotides (shown as "X").
[0310] In addition to the benefits cited above, the use of a
composite design for the oligonucleotides which form the cleavage
structure allows more latitude in the design of the reaction
conditions for performing the Invader.TM.-directed cleavage assay.
When a longer probe (e.g., 16-25 nt), as described in section III
above, is used for detection in reactions that are performed at
temperatures below the T.sub.m of that probe, the cleavage of the
probe may play a significant role in destabilizing the duplex of
which it is a part, thus allowing turnover and reuse of the
recognition site on the target nucleic acid. In contrast, with
miniprobes, reaction temperatures that are at or above the T.sub.m
of the probe mean that the probe molecules are hybridizing and
releasing from the target quite rapidly even without cleavage of
the probe. When an upstream Invader.TM. oligonucleotide and a
cleavage means are provided the miniprobe will be specifically
cleaved, but the cleavage will not be necessary to the turnover of
the miniprobe. If a long probe (e.g., 16-25 nt) were to be used in
this way the temperatures required to achieve this state would be
quite high, around 65 to 70.degree. C. for a 25-mer of average base
composition. Requiring the use of such elevated temperatures limits
the choice of cleavage agents to those that are very thermostable,
and may contribute to background in the reactions, depending of the
means of detection, through thermal degradation of the probe
oligonucleotides. Thus, the shorter probes are preferable for use
in this way.
[0311] The miniprobe of the present invention may vary in size
depending on the desired application. In one embodiment, the probe
may be relatively short compared to a standard probe (e.g., 16-25
nt), in the range of 6 to 10 nucleotides. When such a short probe
is used reaction conditions can be chosen that prevent
hybridization of the miniprobe in the absence of the stacker
oligonucleotide. In this way a short probe can be made to assume
the statistical specificity and selectivity of a longer sequence.
In the event of a perturbation in the cooperative binding of the
miniprobe and stacker nucleic acids, as might be caused by a
mismatch within the short sequence (i.e., region "Z" which is the
region of the miniprobe which does not overlap with the invader) or
at the junction between the contiguous duplexes, this cooperativity
can be lost, dramatically reducing the stability of the shorter
oligonucleotide (i.e., the miniprobe), and thus reducing the level
of cleaved product in the assay of the present invention.
[0312] It is also contemplated that probes of intermediate size may
be used. Such probes, in the 11 to 15 nucleotide range, may blend
some of the features associated with the longer probes as
originally described, these features including the ability to
hybridize and be cleaved absent the help of a stacker
oligonucleotide. At temperatures below the expected T.sub.m of such
probes, the mechanisms of turnover may be as discussed above for
probes in the 20 nt range, and be dependent on the removal of the
sequence in the `X` region for destabilization and cycling.
[0313] The mid-range probes may also be used at elevated
temperatures, at or above their expected T.sub.m, to allow melting
rather than cleavage to promote probe turnover. In contrast to the
longer probes described above, however, the temperatures required
to allow the use of such a thermally driven turnover are much lower
(about 40 to 60.degree. C.), thus preserving both the cleavage
means and the nucleic acids in the reaction from thermal
degradation. In this way, the mid-range probes may perform in some
instances like the miniprobes described above. In a further
similarity to the miniprobes, the accumulation of cleavage signal
from a mid-range probe may be helped under some reaction conditions
by the presence of a stacker.
[0314] To summarize, a standard long probe usually does not benefit
from the presence of a stacker oligonucleotide downstream (the
exception being cases where such an oligonucleotide may also
disrupt structures in the target nucleic acid that interfere with
the probe binding), and it is usually used in conditions requiring
several nucleotides to be removed to allow the oligonucleotide to
release from the target efficiently.
[0315] The miniprobe is very short and performs optimally in the
presence of a downstream stacker oligonucleotide. The miniprobes
are well suited to reactions conditions that use the temperature of
the reaction to drive rapid exchange of the probes on the target
regardeless of whether any bases have been cleaved. In reactions
with sufficient amount of the cleavage means, the probes that do
bind will be rapidly cleaved before they melt off.
[0316] The mid-range or midiprobe combines features of these probes
and can be used in reactions like those designed long probes, with
longer regions of overlap ("X" regions) to drive probe turnover at
lower temperature. In a preferred embodiment, the midrange probes
are used at temperatures sufficiently high that the probes are
hybridizing to the target and releasing rapidly regardless of
cleavage. This is known to be the behavior of oligonucleotides at
or near their melting temperature. This mode of turnover is more
similar to that used with miniprobe/stacker combinations than with
long probes. The mid-range probe may have enhanced performance in
the presence of a stacker under some circumstances. For example,
with a probe in the lower end of the mid-range, e.g., 11 nt, or one
with exceptional A/T content, in a reaction performed well in
excess of the T.sub.m of the probe (e.g., >10.degree. C. above)
the presence of a stacker would be likely to enhance the
performance of the probe, while at a more moderate temperature the
probe may be indifferent to a stacker.
[0317] The distinctions between the mini-, midi- (i.e., mid-range)
and long probes are not contemplated to be inflexible and based
only on length. The performance of any given probe may vary with
its specific sequence, the choice of solution conditions, the
choice of temperature and the selected cleavage means.
[0318] It is shown in Example 18 that the assemblage of
oligonucleotides that comprises the cleavage structure of the
present invention is sensitive to mismatches between the probe and
the target. The site of the mismatch used in Ex. 18 provides one
example and is not intended to be a limitation in location of a
mismatch affecting cleavage. It is also contemplated that a
mismatch between the Invader.TM. oligonucleotide and the target may
be used to distinguish related target sequences. In the
3-oligonucleotide system, comprising an Invader.TM., a probe and a
stacker oligonucleotide, it is contemplated that mismatches may be
located within any of the regions of duplex formed between these
oligonucleotides and the target sequence. In a preferred
embodiment, a mismatch to be detected is located in the probe. In a
particularly preferred embodiment, the mismatch is in the probe, at
the basepair immediately upstream (i.e., 5') of the site that is
cleaved when the probe is not mismatched to the target.
[0319] In another preferred embodiment, a mismatch to be detected
is located within the region `Z` defined by the hybridization of a
miniprobe. In a particularly preferred embodiment, the mismatch is
in the miniprobe, at the basepair immediately upstream (i.e., 5')
of the site that is cleaved when the miniprobe is not mismatched to
the target.
[0320] It is also contemplated that different sequences may be
detected in a single reaction. Probes specific for the different
sequences may be differently labeled. For example, the probes may
have different dyes or other detectable moieties, different
lengths, or they may have differences in net charges of the
products after cleavage. When differently labeled in one of these
ways, the contribution of each specific target sequence to final
product can be tallied. This has application in detecting the
quantities of different versions of a gene within a mixture.
Different genes in a mixture to be detected and quantified may be
wild type and mutant genes, e.g., as may be found in a tumor sample
(e.g., a biopsy). In this embodiment, one might design the probes
to precisely the same site, but one to match the wild-type sequence
and one to match the mutant. Quantitative detection of the products
of cleavage from a reaction performed for a set amount of time will
reveal the ratio of the two genes in the mixture. Such analysis may
also be performed on unrelated genes in a mixture. This type of
analysis is not intended to be limited to two genes. Many variants
within a mixture may be similarly measured.
[0321] Alternatively, different sites on a single gene may be
monitored and quantified to verify the measurement of that gene. In
this embodiment, the signal from each probe would be expected to be
the same.
[0322] It is also contemplated that multiple probes may be used
that are not differently labeled, such that the aggregate signal is
measured. This may be desirable when using many probes designed to
detect a single gene to boost the signal from that gene. This
configuration may also be used for detecting unrelated sequences
within a mix. For example, in blood banking it is desirable to know
if any one of a host of infectious agents is present in a sample of
blood. Because the blood is discarded regardless of which agent is
present, different signals on the probes would not be required in
such an application of the present invention, and may actually be
undesirable for reasons of confidentiality.
[0323] Just as described for the two-oligonucleotide system, above,
the specificity of the detection reaction will be influenced by the
aggregate length of the target nucleic acid sequences involved in
the hybridization of the complete set of the detection
oligonucleotides. For example, there may be applications in which
it is desirable to detect a single region within a complex genome.
In such a case the set of oligonucleotides may be chosen to require
accurate recognition by hybridization of a longer segment of a
target nucleic acid, often in the range of 20 to 40 nucleotides. In
other instances it may be desirable to have the set of
oligonucleotides interact with multiple sites within a target
sample. In these cases one approach would be to use a set of
oligonucleotides that recognize a smaller, and thus statistically
more common, segment of target nucleic acid sequence.
[0324] In one preferred embodiment, the invader and stacker
oligonucleotides may be designed to be maximally stable, so that
they will remain bound to the target sequence for extended periods
during the reaction. This may be accomplished through any one of a
number of measures well known to those skilled in the art, such as
adding extra hybridizing sequences to the length of the
oligonucleotide (up to about 50 nts in total length), or by using
residues with reduced negative charge, such as phosphorothioates or
peptide-nucleic acid residues, so that the complementary strands do
not repel each other to degree that natural strands do. Such
modifications may also serve to make these flanking
oligonucleotides resistant to contaminating nucleases, thus further
ensuring their continued presence on the target strand during the
course of the reaction. In addition, the Invaders and stacker
oligonucleotides may be covalently attached to the target (e.g.,
through the use of psoralen cross-linking).
[0325] The use of the reaction temperatures at or near the T.sub.m
of the probe oligonucleotide, rather thatn the used of cleavage, to
drive the turnover of the probe oligonucleotide in these detection
reactions means that the amount of the probe oligonucleotide
cleaved off may be substantially reduced without adversely
affecting the turnover rate. It has been determined that the
relationship between the 3' end of the upstream oligonucleotide and
the desired site of cleavage on the probe must be carefully
designed. It is known that the preferred site of cleavage for the
types of structure specific endonucleases employed herein is one
basepair into a duplex (Lyamichev et al., supra). It was previously
believed that the presence of an upstream oligonucleotide or primer
allowed the cleavage site to be shifted away from this preferred
site, into the single stranded region of the 5' arm (Lyamichev et
al., supra and U.S. Pat. No. 5,422,253). In contrast to this
previously proposed mechanism, and while not limiting the present
invention to any particular mechanism, it is believed that the
nucleotide immediately 5', or upstream of the cleavage site on the
probe (including miniprobe and mid-range probes) must be able to
basepair with the target for efficient cleavage to occur. In the
case of the present invention, this would be the nucleotide in the
probe sequence immediately upstream of the intended cleavage site.
In addition, as described herein, it has been observed that in
order to direct cleavage to that same site in the probe, the
upstream oligonucleotide must have its 3' base (i.e., nt)
immediately upstream of the the intended cleavage site of the
probe. This places the 3' terminal nucleotide of the upstream
oligonucleotide and the base of the probe oligonucleotide 5' of the
cleavage site in competition for pairing with the corresponding
nucleotide of the target strand.
[0326] To examine the outcome of this competition, i.e. which base
is paired during a successful cleavage event, substitutions were
made in the probe and invader oligonucleotides such that either the
probe or the Invader.TM. oligonucleotide were mismatched with the
target sequence at this position. The effects of both arrangements
on the rates of cleavage were examined. When the Invader.TM.
oligonucleotide is unpaired at the 3' end, the rate of cleavage was
not reduced. If this base was removed, however, the cleavage site
was shifted upstream of the intended site. In contrast, if the
probe oligonucleotide was not base-paired to the target just
upstream of the site to which the Invader.TM. oligonucleotide was
directing cleavage, the rate of cleavage was dramatically reduced,
suggesting that when a competition exists, the probe
oligonucleotide was the molecule to be base-paired in this
position.
[0327] It appears that the 3' end of the upstream invader
oligonucleotide is unpaired during cleavage, and yet is required
for accurate positioning of the cleavage. To examine which part(s)
of the 3' terminal nucleotide are required for the positioning of
cleavage, Invader.TM. oligonucleotides were designed that
terminated on this end with nucleotides that were altered in a
variety of ways. Sugars examined included 2' deoxyribose with a 3'
phosphate group, a dideoxyribose, 3' deoxyribose, 2' O-methyl
ribose, arabinose and arabinose with a 3' phosphate. Abasic ribose,
with and without 3' phosphate were tested. Synthetic "universal"
bases such at 3-nitropyrrole and 5-nitroindole on ribose sugars
were tested. Finally, a base-like aromatic ring structure,
acridine, linked to the 3' end the previous nucleotide without a
sugar group was tested. The results obtained support the conclusion
that the aromatic ring of the base (at the 3' end of the invader
oligonuceotide) is the required moiety for accomplishing the
direction of cleavage to the desired site within the downstream
probe.
VII. Signal Enhancement by Tailing of Reaction Products in the
Invader.TM.-Directed Cleavage Assay
[0328] It has been determined that when oligonucleotide probes are
used in cleavage detection assays at elevated temperature, some
fraction of the truncated probes will have been shortened by
nonspecific thermal degradation, and that such breakage products
can make the analysis of the target-specific cleavage data more
difficult. Background cleavage such as this can, when not resolved
from specific cleavage products, reduce the accuracy of
quantitation of target nucleic acids based on the amount of
accumulated product in a set timeframe. One means of distinguishing
the specific from the nonspecific products is disclosed above, and
is based on partitioning the products of these reactions by
differences in the net charges carried by the different molecular
species in the reaction. As was noted in that discussion, the
thermal breakage products usually retain 3' phosphates after
breakage, while the enzyme-cleaved products do not. The two
negative charges on the phosphate facilitate charge-based partition
of the products.
[0329] The absence of a 3' phosphate on the desired subset of the
probe fragments may be used to advantage in enzymatic assays as
well. Nucleic acid polymerases, both non-templated (e.g., terminal
deoxynucleotidyl transferase, polyA polymerase) and
template-dependent (e.g., Pol I-type DNA polymerases), require an
available 3' hydroxyl by which to attach further nucleotides. This
enzymatic selection of 3' end structure may be used as an effective
means of partitioning specific from non-specific products.
[0330] In addition to the benefits of the partitioning described
above, the addition of nucleotides to the end of the specific
product of an invader-specific cleavage offers an opportunity to
either add label to the products, to add capturable tails to
facilitate solid-support based readout systems, or to do both of
these things at the same time. Some possible embodiments of this
concept are illustrated in FIG. 67.
[0331] In FIG. 67, an Invader.TM. cleavage struture comprising an
Invader.TM. oligonuclotide containing a blocked or non-extendible
3' end (e.g., a 3' dideoxynucleotide) and a probe oligonucleotide
containing a blocked or non-extendable 3' end (the open circle at
the 3' end of the oligonucleotides represents a non-extendible
nucleotide) and a target nucleic acid is shown; the probe
oligonucleotide may contain a 5' end label such as a biotin or a
fluorescein (indicated by the stars) label (cleavage structures
which employ a 5' biotin-labeled probe or a 5' fluorescein-labeled
probe are shown below the large diagram of the cleavage structure
to the left and the right, respectively). Following, cleavage of
the probe (the site of cleavage is indicated by the large
arrowhead), the cleaved biotin-labeled probe is extended using a
template-independent polymerase (e.g., TdT) and fluoresceinated
nucleotide triphosphates. The fluorescein tailed cleaved probe
molecule is then captured by binding via its 5' biotin label to
streptavidin and the fluroescence is then measured. Alternatively,
following, cleavage of a 5'-fluoresceinated probe, the cleaved
probe is extended using a template-independent polymerase (e.g.,
TdT) and DATP. The polyadenylated (A-tailed) cleaved probe molecule
is then captured by binding via the polyA tail to oligo dT attached
to a solid support.
[0332] The examples described in FIG. 66 are based on the use of
TdT to tail the specific products of Invader.TM.-directed cleavage.
The description of the use of this particular enzyme is presented
by way of example and is not intended as a limitation (indeed, when
probe oligos comprising RNA are employed, cleaved RNA probes may be
extended using polyA polymerase). It is contemplated that an assay
of this type could be configured to use a template-dependent
polymerase, as described above. While this would require the
presence of a suitable copy template distinct from the target
nucleic acid, on which the truncated oligonucleotide could prime
synthesis, it can be envisaged that a probe which before cleavage
would be unextendible, due to either mismatch or modification of
the 3' end, could be activated as a primer when cleaved by an
invader directed cleavage. A template directed tailing reaction
also has the advantage of allowing greater selection and control of
the nucleotides incorporated.
[0333] The use of nontemplated tailing does not require the
presence of any additional nucleic acids in the detection reaction,
avoiding one step of assay development and troubleshooting. In
addition, the use of non templated synthesis eliminated the step of
hybridization, potentially speeding up the assay. Furthermore, the
TdT enzyme is fast, able to add at least >700 nucleotides to
substrate oligonucleotides in a 15 minute reaction.
[0334] As mentioned above, the tails added can be used in a number
of ways. It can be used as a straight-forward way of adding labeled
moieties to the cleavage product to increase signal from each
cleavage event. Such a reaction is depicted in the left side of
FIG. 66. The labeled moieties may be anything that can, when
attached to a nucleotide, be added by the tailing enzyme, such as
dye molecules, haptens such as digoxigenin, or other binding groups
such as biotin.
[0335] In a preferred embodiment the assay includes a means of
specifically capturing or partitioning the tailed invader-directed
cleavage products in the mixture. It can be seen that target
nucleic acids in the mixture may be tailed during the reaction. If
a label is added, it is desirable to partition the tailed
invader-directed cleavage products from these other labeled
molecules to avoid background in the results. This is easily done
if only the cleavage product is capable of being captured. For
example, consider a cleavage assay of the present invention in
which the probe used has a biotin on the 5' end and is blocked from
extension on the 3' end, and in which a dye is added during
tailing. Consider further that the products are to be captured onto
a support via the biotin moeity, and the captured dye measured to
assess the presence of the target nucleic acid. When the label is
added by tailing, only the specifically cleaved probes will be
labeled. The residual uncut probes can still bind in the final
capture step, but they will not contribute to the signal. In the
same reaction, nicks and cuts in the target nucleic acid may be
tailed by the enzyme, and thus become dye labeled. In the final
capture these labeled targets will not bind to the support and
thus, though labeled, they will not contribute to the signal. If
the final specific product is considered to consist of two
portions, the probe-derived portion and the tail portion, can be
seen from this discussion that it is particularly preferred that
when the probe-derived portion is used for specific capture,
whether by hybridization, biotin/streptavidin, or other method,
that the label be associated with the tail portion. Conversely, if
a label is attached to the probe-derived portion, then the tail
portion may be made suitable for capture, as depicted on the right
side of FIG. 66. Tails may be captured in a number of ways,
including hybridization, biotin incorporation with streptavidin
capture, or by virtue if the fact that the longer molecules bind
more predictably and efficiently to a number of nucleic acid
minding matrices, such as nitrocellulose, nylon, or glass, in
membrane, paper, resin, or other form. While not required for this
assay, this separation of functions allows effective exclusion from
signal of both unreacted probe and tailed target nucleic acid.
[0336] In addition to the supports decribed above, the tailed
products may be captured onto any support that contains a suitable
capture moiety. For example, biotinylated products are generally
captured with avidin-treated surfaces. These avidin surfaces may be
in microtitre plate wells, on beads, on dipsticks, to name just a
few of the possibilities. Such surfaces can also be modified to
contain specific oligonucleotides, allowing capture of product by
hybridization. Capture surfaces as described here are generally
known to those skilled in the art and include nitrocellulose
dipsticks (e.g., GeneComb, BioRad, Hercules, Calif.).
VIII. Improved Enzymes for Use in Invader.TM.-Directed Cleavage
Reactions
[0337] A cleavage structure is defined herein as a structure which
is formed by the interaction of a probe oligonucleotide and a
target nucleic acid to form a duplex, the resulting structure being
cleavable by a cleavage means, including but not limited to an
enzyme. The cleavage structure is further defined as a substrate
for specific cleavage by the cleavage means in contrast to a
nucleic acid molecule which is a substrate for nonspecific cleavage
by agents such as phosphodiesterases. Examples of some possible
cleavage structures are shown in FIG. 16. In considering
improvements to enzymatic cleavage means, one may consider the
action of said enzymes on any of these structures, and on any other
structures that fall within the definition of a cleavage structure.
The cleavage sites indicated on the structures in FIG. 16 are
presented by way of example. Specific cleavage at any site within
such a structure is contemplated.
[0338] Improvements in an enzyme may be an increased or decreased
rate of cleavage of one or more types of structures. Improvements
may also result in more or fewer sites of cleavage on one or more
of said cleavage structures. In developing a library of new
structure-specific nucleases for use in nucleic acid cleavage
assays, improvements may have many different embodiments, each
related to the specific substrate structure used in a particular
assay.
[0339] As an example, one embodiment of the Invader.TM.-directed
cleavage assay of the present invention may be considered. In the
Invader.TM. directed cleavage assay, the accumulation of cleaved
material is influenced by several features of the enzyme behavior.
Not surprisingly, the turnover rate, or the number of structures
that can be cleaved by a single enzyme molecule in a set amount of
time, is very important in determining the amount of material
processed during the course of an assay reaction. If an enzyme
takes a long time to recognize a substrate (e.g., if it is
presented with a less-than-optimal structure), or if it takes a
long time to execute cleavage, the rate of product accumulation is
lower than if these steps proceeded quickly. If these steps are
quick, yet the enzyme "holds on" to the cleaved structure, and does
not immediately proceed to another uncut structure, the rate will
be negatively affected.
[0340] Enzyme turnover is not the only way in which enzyme behavior
can negatively affect the rate of accumulation of product. When the
means used to visualize or measure product is specific for a
precisely defined product, products that deviate from that
definition may escape detection, and thus the rate of product
accumulation may appear to be lower than it is. For example, if one
had a sensitive detector for trinucleotides that could not see di-
or tetranucleotides, or any sized oligonucleotide other that 3
residues, in the iIvader.TM.-directed cleavage assay of the present
invention any errant cleavage would reduce the detectable signal
proportionally. It can be seen from the cleavage data presented
here that, while there is usually one site within a probe that is
favored for cleavage, there are often products that arise from
cleavage one or more nucleotides away from the primary cleavage
site. These are products that are target dependent, and are thus
not non-specific background. Nevertheless, if a subsequent
visualization system can detect only the primary product, these
represent a loss of signal. One example of such a selective
visualization system is the charge reversal readout presented
herein, in which the balance of positive and negative charges
determines the behavior of the products. In such a system the
presence of an extra nucleotide or the absence of an expected
nucleotide can excluded a legitimate cleavage product from ultimate
detection by leaving that product with the wrong balance of charge.
It can be easily seen that any assay that can sensitively
distinguish the nucleotide content of an oligonucleotide, such as
standard stringent hybridization, suffers in sensitivity when some
fraction of the legitimate product is not eligible for successful
detection by that assay.
[0341] These discussions suggest two highly desirable traits in any
enzyme to be used in the method of the present invention. First,
the more rapidly the enzyme executes an entire cleavage reaction,
including recognition, cleavage and release, the more signal it may
potentially created in the invader-directed cleavage assay. Second,
the more successful an enzyme is at focusing on a single cleavage
site within a structure, the more of the cleavage product can be
successfully detected in a selective read-out. The rationale cited
above for making improvements in enzymes to be used in the
Invader.TM.-directed cleavage assay are meant to serve as an
example of one direction in which improvements might be sought, but
not as a limit on either the nature or the applications of improved
enzyme activities. As another direction of activity change that
would be appropriately considered improvement, the DNAP-associated
5' nucleases may be used as an example. In creating some of the
polymerase-deficient 5' nucleases described herein it was found
that the those that were created by deletion of substantial
portions of the polymerase domain, as depicted in FIG. 4, assumed
activities that were weak or absent in the parent proteins. These
activities included the ability to cleave the non-forked structure
shown in FIG. 16D, a greatly enhanced ability to exonucleolytically
remove nucleotides from the 5' ends of duplexed strands, and a
nascent ability to cleave circular molecules without benefit of a
free 5' end. These features have contributed to the development of
detection assays such as the one depicted in FIG. 1A.
[0342] In addition to the 5' nucleases derived from DNA
polymerases, the present invention also contemplates the use of
structure-specific nucleases that are not derived from DNA
polymerases. For example, a class of eukaryotic and archaebacterial
endonucleases have been identified which have a similar substrate
specificity to 5' nucleases of Pol I-type DNA polymerases. These
are the FENI (Flap EndoNuclease), RAD2, and XPG (Xeroderma
Pigmentosa-complementation group G) proteins. These proteins are
involved in DNA repair, and have been shown to favor the cleavage
of structures that resemble a 5' arm that has been displaced by an
extending primer during polymerization, similar to the model
depicted in FIG. 16B. Similar DNA repair enzymes have been isolated
from single cell and higher eukaryotes and from archaea, and there
are related DNA repair proteins in eubacteria. Similar 5' nucleases
have also be associated with bacteriophage such as T5 and T7.
[0343] Recently, the 3-dimensional structures of DNAPTaq and T5
phage 5'-exonuclease (FIG. 69) were determined by X-ray diffraction
[Kim et al. (1995) Nature 376:612 and Ceska et al. (1995) Nature
382:90). The two enzymes have very similar 3-dimensional structures
despite limited amino acid sequence similarity. The most striking
feature of the T5 5'-exonuclease structure is the existence of a
triangular hole formed by the active site of the protein and two
alpha helices (FIG. 69). This same region of DNAPTaq is disordered
in the crystal structure, indicating that this region is flexible,
and thus is not shown in the published 3-dimensional structure.
However, the 5' nuclease domain of DNAPTaq is likely to have the
same structure, based its overall 3-dimensional similarity to T5
5'-exonuclease, and that the amino acids in the disordered region
of the DNAPTaq protein are those associated with alpha helix
formation. The existence of such a hole or groove in the 5'
nuclease domain of DNAPTaq was predicted based on its substrate
specificity [Lyamichev et al., supra].
[0344] It has been suggested that the 5' arm of a cleavage
structure must thread through the helical arch described above to
position said structure correctly for cleavage (Ceska et al.,
supra). One of the modifications of 5' nucleases described herein
opened up the helical arch portion of the protein to allow improved
cleavage of structures that cut poorly or not at all (e.g.,
structures on circular DNA targets that would preclude such
threading of a 5' arm). The gene construct that was chosen as a
model to test this approach was the one called Cleavase.RTM. BN,
which was derived from DNAPTaq but does not contain the polymerase
domainn (Ex. 2). It comprises the entire 5' nuclease domain of DNAP
Taq, and thus should be very close in structure to the T5 5'
exonuclease. This 5' nuclease was chosen to demonstrate the
principle of such a physical modification on proteins of this type.
The arch-opening modification of the present invention is not
intended to be limited to the 5' nuclease domains of DNA
polymerases, and is contemplated for use on any structure-specific
nuclease which includes such an aperture as a limitation on
cleavage activity.
[0345] The opening of the helical arch was accomplished by
insertion of a protease site in the arch. This allowed
post-translational digestion of the expressed protein with the
appropriate protease to open the arch at its apex. Proteases of
this type recognize short stretches of specific amino acid
sequence. Such proteases include thrombin and factor Xa. Cleavage
of a protein with such a protease depends on both the presence of
that site in the amino acid sequence of the protein and the
accessibility of that site on the folded intact protein. Even with
a crystal structure it can be difficult to predict the
susceptibility of any particular region of a protein to protease
cleavage. Absent a crystal structure it must be determined
empirically.
[0346] In selecting a protease for a site-specific cleavage of a
protein that has been modified to contain a protease cleavage site,
a first step is to test the unmodified protein for cleavage at
alternative sites. For example, DNAPTaq and Cleavase.RTM. BN
nuclease were both incubated under protease cleavage conditions
with factor Xa and thrombin proteases. Both nuclease proteins were
cut with factor Xa within the 5' nuclease domain, but neither
nuclease was digested with large amounts of thrombin. Thus,
thrombin was chosen for initial tests on opening the arch of the
Cleavase.RTM. BN enzyme.
[0347] In the protease/Cleavase.RTM. modifications described herein
the factor Xa protease cleaved strongly in an unacceptable position
in the unmodified nuclease protein, in a region likely to
compromise the activity of the end product. Other unmodified
nucleases contemplated herein may not be sensitive to the factor
Xa, but may be sensitive to thrombin or other such proteases.
Alternatively, they may be sensitive to these or other such
proteases at sites that are immaterial to the function of the
nuclease sought to be modified. In approaching any protein for
modification by addition of a protease cleavage site, the
unmodified protein should be tested with the proteases under
consideration to determine which proteases give acceptable levels
of cleavage in other regions.
[0348] Working with the cloned segment of DNAPTaq from which the
Cleavase.RTM. BN protein is expressed, nucleotides encoding a
thrombin cleavage site were introduced in-frame near the sequence
encoding amino acid 90 of the nuclease gene. This position was
determined to be at or near the apex of the helical arch by
reference to both the 3-dimensional structure of DNAPTaq, and the
structure of T5 5' exonuclease.
[0349] The encoded amino acid sequence, LVPRGS, was inserted into
the apex of the helical arch by site-directed mutagenesis of the
nuclease gene. The proline (P) in the thrombin cleavage site was
positioned to replace a proline normally in this position in
Cleavase.RTM. BN because proline is an alpha helix-breaking amino
acid, and may be important for the 3-dimensional structure of this
arch. This construct was expressed, purified and then digested with
thrombin. The digested enzyme was tested for its ability to cleave
a target nucleic acid, bacteriophage M13 genomic DNA, that does not
provide free 5' ends to facilitate cleavage by the threading
model.
[0350] While the helical arch in this nuclease was opened by
protease cleavage, it is contemplated that a number of other
techniques could be used to achieve the same end. For example, the
nucleotide sequence could be rearranged such that, upon expression,
the resulting protein would be configured so that the top of the
helical arch (amino acid 90) would be at the amino terminus of the
protein, the natural carboxyl and amino termini of the protein
sequence would be joined, and the new carboxyl terminus would lie
at natural amino acid 89. This approach has the benefit that no
foreign sequences are introduced and the enzyme is a single amino
acid chain, and thus may be more stable that the cleaved 5'
nuclease. In the crystal structure of DNAPTaq, the amino and
carboxyl termini of the 5'-exonuclease domain lie in close
proximity to each other, which suggests that the ends may be
directly joined without the use of a flexible linker peptide
sequence as is sometimes necessary. Such a rearrangement of the
gene, with subsequent cloning and expression could be accomplished
by standard PCR recombination and cloning techniques known to those
skilled in the art.
[0351] The present invention also contemplates the use of nucleases
isolated from a organisms that grow under a variety of conditions.
The genes for the FEN-1/XPG class of enzymes are found in organisms
ranging from bacteriophage to humans to the extreme thermophiles of
Kingdom Archaea. For assays in which high temperature is to be
used, it is contemplated that enzymes isolated from extreme
thermophiles may exhibit the thermostability required of such an
assay. For assays in which it might be desirable to have peak
enzyme activity at moderate temperature or in which it might be
desirable to destroy the enzyme with elevated temperature, those
enzymes from organisms that favor moderate temperatures for growth
may be of particular value.
[0352] An alignment of a collection of FEN-1 proteins sequenced by
others is shown in FIGS. 70A-E. It can be seen from this alignment
that there are some regions of conservation in this class of
proteins, suggesting that they are related in function, and
possibly in structure. Regions of similarity at the amino acid
sequence level can be used to design primers for in vitro
amplification (PCR) by a process of back translating the amino acid
sequence to the possible nucleic acid sequences, then choosing
primers with the fewest possible variations within the sequences.
These can be used in low stringency PCR to search for related DNA
sequences. This approach permits the amplification of DNA encoding
a FEN-1 nuclease without advance knowledge of the actual DNA
sequence.
[0353] It can also be seen from this alignment that there are
regions in the sequences that are not completely conserved. The
degree of difference observed suggests that the proteins may have
subtle or distinct differences is substrate specificity. In other
words, they may have different levels of cleavage activity on the
cleavage structures of the present invention. When a particular
structure is cleaved at a higher rate than the others, this is
referred to a preferred substrate, while a structure that is
cleaved slowly is considered a less preferred substrate. The
designation of preferred or less preferred substrates in this
context is not intended to be a limitation of the present
invention. It is contemplated that some embodiments the present
invention will make use of the interactions of an enzyme with a
less preferred substrate. Candidate enzymes are tested for
suitability in the cleavage assays of the present invention using
the assays described below.
[0354] 1. Structure Specific Nuclease Assay
[0355] Testing candidate nucleases for structure-specific
activities in these assays is done in much the same way as
described for testing modified DNA polymerases in Example 2, but
with the use of a different library of model structures. In
addition to assessing the enzyme performance in primer-independent
and primer-directed cleavage, a set of synthetic hairpins are used
to examine the length of duplex downstream of the cleavage site
preferred by the enzyme.
[0356] The FEN-1 and XPG 5' nucleases used in the present invention
must be tested for activity in the assays in which they are
intended to be used, including but not limited to the
Invader.TM.-directed cleavage detection assay of the present
invention and the CFLP.RTM. method of characterizing nucleic acids
(the CFLP.RTM. method is described in co-pending application Ser.
Nos. 08/337,164, 08/402,601, 08/484,956 and 08/520,946; the
disclosures of these applications are incorporated herein by
reference). The Invader.TM. assay uses a mode of cleavage that has
been termed "primer directed" of "primer dependent" to reflect the
influence of the an oligonucleotide hybridized to the target
nucleic acid upstream of the cleavage site. In contrast, the
CFLP.RTM. reaction is based on the cleavage of folded structure, or
hairpins, within the target nucleic acid, in the absence of any
hybridized oligonucleotide. The tests described herein are not
intended to be limited to the analysis of nucleases with any
particular site of cleavage or mode of recognition of substrate
structures. It is contemplated that enzymes may be described as 3'
nucleases, utilizing the 3' end as a reference point to recognize
structures, or may have a yet a different mode of recognition.
Further, the use of the term 5' nucleases is not intended to limit
consideration to enzymes that cleave the cleavage structures at any
particular site. It refers to a general class of enzymes that
require some reference or access to a 5' end to effect cleavage of
a structure.
[0357] A set of model cleavage structures have been created to
allow the cleavage ability of unknown enzymes on such structures to
be assessed. Each of the model structures is constructed of one or
more synthetic oligonucleotides made by standard DNA synthesis
chemistry. Examples of such synthetic model substrate structures
are shown in FIGS. 30 and 70. These are intended only to represent
the general folded configuration desirable is such test structures.
While a sequence that would assume such a structure is indicated in
the figures, there are numerous other sequence arrangements of
nucleotides that would be expected to fold in such ways. The
essential features to be designed into a set of oligonucleotides to
perform the tests described herein are the presence or absence of a
sufficiently long 3' arm to allow hybridization of an additional
nucleic acid to test cleavage in a "primer-directed" mode, and the
length of the duplex region. In the set depicted in FIG. 71, the
duplex lengths of the S-33 and the 11-8-0 structures are 0.12 and 8
basepairs, respectively. This difference in length in the test
molecules facilitates detection of discrimination by the candidate
nuclease between longer and shorter duplexes. Additions to this
series expanding the range of duplex molecules presented to the
enzymes, both shorter and longer, may be used. The use of a
stabilizing DNA tetraloop [Antao et al. (1991) Nucl. Acids Res.
19:5901] or triloop [Hiraro et al. (1994) Nuc. Acids Res. 22:576]
at the closed end of the duplex helps ensure formation of the
expected structure by the oligonucleotide.
[0358] The model substrate for testing primer directed cleavage,
the "S-60 hairpin" (SEQ ID NO:40) is described in Example 11. In
the absence of a primer this hairpin is usually cleaved to release
5' arm fragments of 18 and 19 nucleotides length. An
oligonucleotide, termed P-14 (5'-CGAGAGACCACGCT-3'), that extends
to the base of the duplex when hybridized to the 3' arm of the S-60
hairpin gives cleavage products of the same size, but at a higher
rate of cleavage.
[0359] To test invasive cleavage a different primer is used, termed
P-15 (5'-CGAGAGACCACGCTG-3'). In a successful invasive cleavage the
presence of this primer shifts the site of cleavage of S-60 into
the duplex region, usually releasing products of 21 and 22
nucleotides length.
[0360] The S-60 hairpin may also be used to test the effects of
modifications of the cleavage structure on either primer-directed
or invasive cleavage. Such modifications include, but are not
limited to, use of mismatches or base analogs in the hairpin duplex
at one, a few or all positions, similar disruptions or
modifications in the duplex between the primer and the 3' arm of
the S-60, chemical or other modifications to one or both ends of
the primer sequence, or attachment of moieties to, or other
modifications of the 5' arm of the structure. In all of the
analyses using the S-60 or a similar hairpin described herein,
activity with and without a primer may be compared using the same
hairpin structure.
[0361] The assembly of these test reactions, including appropriate
amounts of hairpin, primer and candidate nuclease are described in
Example 2. As cited therein, the presence of cleavage products is
indicated by the presence of molecules which migrate at a lower
molecular weight than does the uncleaved test structure. When the
reversal of charge of a label is used the products will carry a
different net charge than the uncleaved material. Any of these
cleavage products indicate that the candidate nuclease has the
desired structure-specific nuclease activity. By "desired
structure-specific nuclease activity" it is meant only that the
candidate nuclease cleaves one or more test molecules. It is not
necessary that the candidate nuclease cleave at any particular rate
or site of cleavage to be considered successful cleavage.
EXPERIMENTAL
[0362] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
[0363] In the disclosure which follows, the following abbreviations
apply: .degree. C. (degrees Centigrade); g (gravitational field);
vol (volume); w/v (weight to volume); v/v (volume to volume); BSA
(bovine serum albumin); CTAB (cetyltrimethylammonium bromide); HPLC
(high pressure liquid chromatography); DNA (deoxyribonucleic acid);
p (plasmid); .mu.l (microliters); ml (milliliters); .mu.g
(micrograms); pmoles (picomoles); mg (milligrams); M (molar); mM
(milliMolar); .mu.M (microMolar); nm (nanometers); kdal
(kilodaltons); OD (optical density); EDTA (ethylene diamine
tetra-acetic acid); FITC (fluorescein isothiocyanate); SDS (sodium
dodecyl sulfate); NaPO.sub.4 (sodium phosphate);
Tris(tris(hydroxymethyl)-aminomethane); PMSF
(phenylmethylsulfonylfluoride); TBE (Tris-Borate-EDTA, i.e., Tris
buffer titrated with boric acid rather than HCl and containing
EDTA); PBS (phosphate buffered saline); PPBS (phosphate buffered
saline containing 1 mM PMSF); PAGE (polyacrylamide gel
electrophoresis); Tween (polyoxyethylene-sorbitan); Dynal (Dynal A.
S., Oslo, Norway); Epicentre (Epicentre Technologies, Madison,
Wis.); MJ Research (MJ Research, Watertown, Mass.); National
Biosciences (Plymouth, Minn.); New England Biolabs (Beverly,
Mass.); Novagen (Novagen, Inc., Madison, Wis.); Perkin Elmer
(Norwalk, Conn.); Promega Corp. (Madison, Wis.); Stratagene
(Stratagene Cloning Systems, La Jolla, Calif.); USB (U.S.
Biochemical, Cleveland, Ohio).
Example 1
Characteristics of Native Thermostable DNA Polymerases
[0364] A. 5' Nuclease Activity Of DNAPTaq
[0365] During the polymerase chain reaction (PCR) [Saiki et al.,
Science 239:487 (1988); Mullis and Faloona, Methods in Enzymology
155:335 (1987)], DNAPTaq is able to amplify many, but not all, DNA
sequences. One sequence that cannot be amplified using DNAPTaq is
shown in FIG. 6 (Hairpin structure is SEQ ID NO:15, PRIMERS are SEQ
ID NOS:16-17.) This DNA sequence has the distinguishing
characteristic of being able to fold on itself to form a hairpin
with two single-stranded arms, which correspond to the primers used
in PCR.
[0366] To test whether this failure to amplify is due to the 5'
nuclease activity of the enzyme, we compared the abilities of
DNAPTaq and DNAPStf to amplify this DNA sequence during 30 cycles
of PCR. Synthetic oligonucleotides were obtained from The
Biotechnology Center at the University of Wisconsin-Madison. The
DNAPTaq and DNAPStf were from Perkin Elmer (i.e., Amplitaq.TM. DNA
polymerase and the Stoffel fragment of Amplitaq.TM. DNA
polymerase). The substrate DNA comprised the hairpin structure
shown in FIG. 6 cloned in a double-stranded form into pUC19. The
primers used in the amplification are listed as SEQ ID NOS:16-17.
Primer SEQ ID NO:17 is shown annealed to the 3' arm of the hairpin
structure in FIG. 6. Primer SEQ ID NO:16 is shown as the first 20
nucleotides in bold on the 5' arm of the hairpin in FIG. 6.
[0367] Polymerase chain reactions comprised 1 ng of supercoiled
plasmid target DNA, 5 pmoles of each primer, 40 .mu.M each dNTP,
and 2.5 units of DNAPTaq or DNAPStf, in a 50 .mu.l solution of 10
mM Tris.Cl pH 8.3. The DNAPTaq reactions included 50 mM KCl and 1.5
mM MgCl.sub.2. The temperature profile was 95.degree. C. for 30
sec., 55.degree. C. for 1 min. and 72.degree. C. for 1 min.,
through 30 cycles. Ten percent of each reaction was analyzed by gel
electrophoresis through 6% polyacrylamide (cross-linked 29:1) in a
buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.
[0368] The results are shown in FIG. 7. The expected product was
made by DNAPStf (indicated simply as "S") but not by DNAPTaq
(indicated as "T"). We conclude that the 5' nuclease activity of
DNAPTaq is responsible for the lack of amplification of this DNA
sequence.
[0369] To test whether the 5' unpaired nucleotides in the substrate
region of this structured DNA are removed by DNAPTaq, the fate of
the end-labeled 5' arm during four cycles of PCR was compared using
the same two polymerases (Figure. 8). The hairpin templates, such
as the one described in FIG. 6, were made using DNAPStf and a
.sup.32P-5'-end-labeled primer. The 5'-end of the DNA was released
as a few large fragments by DNAPTaq but not by DNAPStf. The sizes
of these fragments (based on their mobilities) show that they
contain most or all of the unpaired 5' arm of the DNA. Thus,
cleavage occurs at or near the base of the bifurcated duplex. These
released fragments terminate with 3' OH groups, as evidenced by
direct sequence analysis, and the abilities of the fragments to be
extended by terminal deoxynucleotidyl transferase.
[0370] FIGS. 9-11 show the results of experiments designed to
characterize the cleavage reaction catalyzed by DNAPTaq. Unless
otherwise specified, the cleavage reactions comprised 0.01 pmoles
of heat-denatured, end-labeled hairpin DNA (with the unlabeled
complementary strand also present), 1 pmole primer (complementary
to the 3' arm) and 0.5 units of DNAPTaq (estimated to be 0.026
pmoles) in a total volume of 10 .mu.l of 10 mM Tris-Cl, ph 8.5, 50
mM KCl and 1.5 mM MgCl.sub.2. As indicated, some reactions had
different concentrations of KCl, and the precise times and
temperatures used in each experiment are indicated in the
individual figures. The reactions that included a primer used the
one shown in FIG. 6 (SEQ ID NO:17). In some instances, the primer
was extended to the junction site by providing polymerase and
selected nucleotides.
[0371] Reactions were initiated at the final reaction temperature
by the addition of either the MgCl.sub.2 or enzyme. Reactions were
stopped at their incubation temperatures by the addition of 8 .mu.l
of 95% formamide with 20 mM EDTA and 0.05% marker dyes. The T.sub.m
calculations listed were made using the Oligo.TM. primer analysis
software from National Biosciences, Inc. These were determined
using 0.25 .mu.M as the DNA concentration, at either 15 or 65 mM
total salt (the 1.5 mM MgCl.sub.2 in all reactions was given the
value of 15 mM salt for these calculations).
[0372] FIG. 9 is an autoradiogram containing the results of a set
of experiments and conditions on the cleavage site. FIG. 9A is a
determination of reaction components that enable cleavage.
Incubation of 5'-end-labeled hairpin DNA was for 30 minutes at
55.degree. C., with the indicated components. The products were
resolved by denaturing polyacrylamide gel electrophoresis and the
lengths of the products, in nucleotides, are indicated. FIG. 9B
describes the effect of temperature on the site of cleavage in the
absence of added primer. Reactions were incubated in the absence of
KCl for 10 minutes at the indicated temperatures. The lengths of
the products, in nucleotides, are indicated.
[0373] Surprisingly, cleavage by DNAPTaq requires neither a primer
nor dNTPs (see FIG. 9A). Thus, the 5' nuclease activity can be
uncoupled from polymerization. Nuclease activity requires magnesium
ions, though manganese ions can be substituted, albeit with
potential changes in specificity and activity. Neither zinc nor
calcium ions support the cleavage reaction. The reaction occurs
over a broad temperature range, from 25.degree. C. to 85.degree.
C., with the rate of cleavage increasing at higher
temperatures.
[0374] Still referring to FIG. 9, the primer is not elongated in
the absence of added dNTPs. However, the primer influences both the
site and the rate of cleavage of the hairpin. The change in the
site of cleavage (FIG. 9A) apparently results from disruption of a
short duplex formed between the arms of the DNA substrate. In the
absence of primer, the sequences indicated by underlining in FIG. 6
could pair, forming an extended duplex. Cleavage at the end of the
extended duplex would release the 11 nucleotide fragment seen on
the FIG. 9A lanes with no added primer. Addition of excess primer
(FIG. 9A, lanes 3 and 4) or incubation at an elevated temperature
(FIG. 9B) disrupts the short extension of the duplex and results in
a longer 5' arm and, hence, longer cleavage products.
[0375] The location of the 3' end of the primer can influence the
precise site of cleavage. Electrophoretic analysis revealed that in
the absence of primer (FIG. 9B), cleavage occurs at the end of the
substrate duplex (either the extended or shortened form, depending
on the temperature) between the first and second base pairs. When
the primer extends up to the base of the duplex, cleavage also
occurs one nucleotide into the duplex. However, when a gap of four
or six nucleotides exists between the 3' end of the primer and the
substrate duplex, the cleavage site is shifted four to six
nucleotides in the 5' direction.
[0376] FIG. 10 describes the kinetics of cleavage in the presence
(FIG. 10A) or absence (FIG. 10B) of a primer oligonucleotide. The
reactions were run at 55.degree. C. with either 50 mM KCl (FIG.
10A) or 20 mM KCl (FIG. 10B). The reaction products were resolved
by denaturing polyacrylamide gel electrophoresis and the lengths of
the products, in nucleotides, are indicated. "M", indicating a
marker, is a 5' end-labeled 19-nt oligonucleotide. Under these salt
conditions, FIGS. 10A and 10B indicate that the reaction appears to
be about twenty times faster in the presence of primer than in the
absence of primer. This effect on the efficiency may be
attributable to proper alignment and stabilization of the enzyme on
the substrate.
[0377] The relative influence of primer on cleavage rates becomes
much greater when both reactions are run in 50 mM KCl. In the
presence of primer, the rate of cleavage increases with KCl
concentration, up to about 50 mM. However, inhibition of this
reaction in the presence of primer is apparent at 100 mM and is
complete at 150 mM KCl. In contrast, in the absence of primer the
rate is enhanced by concentration of KCl up to 20 mM, but it is
reduced at concentrations above 30 mM. At 50 mM KCl, the reaction
is almost completely inhibited. The inhibition of cleavage by KCl
in the absence of primer is affected by temperature, being more
pronounced at lower temperatures.
[0378] Recognition of the 5' end of the arm to be cut appears to be
an important feature of substrate recognition. Substrates that lack
a free 5' end, such as circular M13 DNA, cannot be cleaved under
any conditions tested. Even with substrates having defined 5' arms,
the rate of cleavage by DNAPTaq is influenced by the length of the
arm. In the presence of primer and 50 mM KCl, cleavage of a 5'
extension that is 27 nucleotides long is essentially complete
within 2 minutes at 55.degree. C. In contrast, cleavages of
molecules with 5' arms of 84 and 188 nucleotides are only about 90%
and 40% complete after 20 minutes. Incubation at higher
temperatures reduces the inhibitory effects of long extensions
indicating that secondary structure in the 5' arm or a heat-labile
structure in the enzyme may inhibit the reaction. A mixing
experiment, run under conditions of substrate excess, shows that
the molecules with long arms do not preferentially tie up the
available enzyme in non-productive complexes. These results may
indicate that the 5' nuclease domain gains access to the cleavage
site at the end of the bifurcated duplex by moving down the 5' arm
from one end to the other. Longer 5' arms would be expected to have
more adventitious secondary structures (particularly when KCl
concentrations are high), which would be likely to impede this
movement.
[0379] Cleavage does not appear to be inhibited by long 3' arms of
either the substrate strand target molecule or pilot nucleic acid,
at least up to 2 kilobases. At the other extreme, 3' arms of the
pilot nucleic acid as short as one nucleotide can support cleavage
in a primer-independent reaction, albeit inefficiently. Fully
paired oligonucleotides do not elicit cleavage of DNA templates
during primer extension.
[0380] The ability of DNAPTaq to cleave molecules even when the
complementary strand contains only one unpaired 3' nucleotide may
be useful in optimizing allele-specific PCR. PCR primers that have
unpaired 3' ends could act as pilot oligonucleotides to direct
selective cleavage of unwanted templates during preincubation of
potential template-primer complexes with DNAPTaq in the absence of
nucleoside triphosphates.
[0381] B. 5' Nuclease Activities of Other DNAPs
[0382] To determine whether other 5' nucleases in other DNAPs would
be suitable for the present invention, an array of enzymes, several
of which were reported in the literature to be free of apparent 5'
nuclease activity, were examined. The ability of these other
enzymes to cleave nucleic acids in a structure-specific manner was
tested using the hairpin substrate shown in FIG. 6 under conditions
reported to be optimal for synthesis by each enzyme.
[0383] DNAPEc1 and DNAP Klenow were obtained from Promega
Corporation; the DNAP of Pyrococcus furious ["Pfu", Bargseid et
al., Strategies 4:34 (1991)] was from Strategene; the DNAP of
Thermococcus litoralis ["Tli", Vent.TM.(exo-), Perler et al., Proc.
Natl. Acad. Sci. USA 89:5577 (1992)] was from New England Biolabs;
the DNAP of Thermus flavus ["Tfl", Kaledin et al., Biokhimiya
46:1576 (1981)] was from Epicentre Technologies; and the DNAP of
Thermus thermophilus ["Tth", Carballeira et al., Biotechniques
9:276 (1990); Myers et al., Biochem. 30:7661 (1991)] was from U.S.
Biochemicals.
[0384] 0.5 units of each DNA polymerase was assayed in a 20 .mu.l
reaction, using either the buffers supplied by the manufacturers
for the primer-dependent reactions, or 10 mM Tris.Cl, pH 8.5, 1.5
mM MgCl.sub.2, and 20 mM KCl. Reaction mixtures were at held
72.degree. C. before the addition of enzyme.
[0385] FIG. 11 is an autoradiogram recording the results of these
tests. FIG. 11A demonstrates reactions of endonucleases of DNAPs of
several thermophilic bacteria. The reactions were incubated at
55.degree. C. for 10 minutes in the presence of primer or at
72.degree. C. for 30 minutes in the absence of primer, and the
products were resolved by denaturing polyacrylamide gel
electrophoresis. The lengths of the products, in nucleotides, are
indicated. FIG. 1B demonstrates endonucleolytic cleavage by the 5'
nuclease of DNAPEc1. The DNAPEc1 and DNAP Klenow reactions were
incubated for 5 minutes at 37.degree. C. Note the light band of
cleavage products of 25 and 11 nucleotides in the DNAPEc1 lanes
(made in the presence and absence of primer, respectively). FIG. 7B
also demonstrates DNAPTaq reactions in the presence (+) or absence
(-) of primer. These reactions were run in 50 mM and 20 mM KCl,
respectively, and were incubated at 55.degree. C. for 10
minutes.
[0386] Referring to FIG. 11A, DNAPs from the eubacteria Thermus
thermophilus and Thermus flavus cleave the substrate at the same
place as DNAPTaq, both in the presence and absence of primer. In
contrast, DNAPs from the archaebacteria Pyrococcus furiosus and
Thermococcus litoralis are unable to cleave the substrates
endonucleolytically. The DNAPs from Pyrococcus furious and
Thermococcus litoralis share little sequence homology with
eubacterial enzymes (Ito et al., Nucl. Acids Res. 19:4045 (1991);
Mathur et al., Nucl. Acids. Res. 19:6952 (1991); see also Perler et
al.). Referring to FIG. 11B, DNAPEc1 also cleaves the substrate,
but the resulting cleavage products are difficult to detect unless
the 3' exonuclease is inhibited. The amino acid sequences of the 5'
nuclease domains of DNAPEc1 and DNAPTaq are about 38% homologous
(Gelfand, supra).
[0387] The 5' nuclease domain of DNAPTaq also shares about 19%
homology with the 5' exonuclease encoded by gene 6 of bacteriophage
T7 [Dunn et al., J. Mol. Biol. 166:477 (1983)]. This nuclease,
which is not covalently attached to a DNAP polymerization domain,
is also able to cleave DNA endonucleolytically, at a site similar
or identical to the site that is cut by the 5' nucleases described
above, in the absence of added primers.
[0388] C. Transcleavage
[0389] The ability of a 5' nuclease to be directed to cleave
efficiently at any specific sequence was demonstrated in the
following experiment. A partially complementary oligonucleotide
termed a "pilot oligonucleotide" was hybridized to sequences at the
desired point of cleavage. The non-complementary part of the pilot
oligonucleotide provided a structure analogous to the 3' arm of the
template (see FIG. 6), whereas the 5' region of the substrate
strand became the 5' arm. A primer was provided by designing the 3'
region of the pilot so that it would fold on itself creating a
short hairpin with a stabilizing tetra-loop [Antao et al., Nucl.
Acids Res. 19:5901 (1991)]. Two pilot oligonucleotides are shown in
FIG. 12A. Oligonucleotides 19-12 (SEQ ID NO:18), 30-12 (SEQ ID
NO:19) and 30-0 (SEQ ID NO:20) are 31, 42 or 30 nucleotides long,
respectively. However, oligonucleotides 19-12 (SEQ ID NO:18) and
34-19 (SEQ ID NO:19) have only 19 and 30 nucleotides, respectively,
that are complementary to different sequences in the substrate
strand. The pilot oligonucleotides are calculated to melt off their
complements at about 50.degree. C. (19-12) and about 75.degree. C.
(30-12). Both pilots have 12 nucleotides at their 3' ends, which
act as 3' arms with base-paired primers attached.
[0390] To demonstrate that cleavage could be directed by a pilot
oligonucleotide, we incubated a single-stranded target DNA with
DNAPTaq in the presence of two potential pilot oligonucleotides.
The transcleavage reactions, where the target and pilot nucleic
acids are not covalently linked, includes 0.01 pmoles of single
end-labeled substrate DNA, 1 unit of DNAPTaq and 5 pmoles of pilot
oligonucleotide in a volume of 20 .mu.l of the same buffers. These
components were combined during a one minute incubation at
95.degree. C., to denature the PCR-generated double-stranded
substrate DNA, and the temperatures of the reactions were then
reduced to their final incubation temperatures. Oligonucleotides
30-12 and 19-12 can hybridize to regions of the substrate DNAs that
are 85 and 27 nucleotides from the 5' end of the targeted
strand.
[0391] FIG. 21 shows the complete 206-mer sequence (SEQ ID NO:32).
The 206-mer was generated by PCR. The M13/pUC 24-mer reverse
sequencing (-48) primer and the M13/pUC sequencing (-47) primer
from New England Biolabs (catalogue nos. 1233 and 1224
respectively) were used (50 pmoles each) with the pGEM3z(f+)
plasmid vector (Promega Corp.) as template (10 ng) containing the
target sequences. The conditions for PCR were as follows: 50 .mu.M
of each dNTP and 2.5 units of Taq DNA polymerase in 100 .mu.l of 20
mM Tris-Cl, pH 8.3, 1.5 mM MgCl.sub.2, 50 mM KCl with 0.05%
Tween-20 and 0.05% NP-40. Reactions were cycled 35 times through
95.degree. C. for 45 seconds, 63.degree. C. for 45 seconds, then
72.degree. C. for 75 seconds. After cycling, reactions were
finished off with an incubation at 72.degree. C. for 5 minutes. The
resulting fragment was purified by electrophoresis through a 6%
polyacrylamide gel (29:1 cross link) in a buffer of 45 mM
Tris-Borate, pH 8.3, 1.4 mM EDTA, visualized by ethidium bromide
staining or autoradiography, excised from the gel, eluted by
passive diffusion, and concentrated by ethanol precipitation.
[0392] Cleavage of the substrate DNA occurred in the presence of
the pilot oligonucleotide 19-12 at 50.degree. C. (FIG. 12B, lanes 1
and 7) but not at 75.degree. C. (lanes 4 and 10). In the presence
of oligonucleotide 30-12 cleavage was observed at both
temperatures. Cleavage did not occur in the absence of added
oligonucleotides (lanes 3, 6 and 12) or at about 80.degree. C. even
though at 50.degree. C. adventitious structures in the substrate
allowed primer-independent cleavage in the absence of KCl (FIG.
12B, lane 9). A non-specific oligonucleotide with no
complementarity to the substrate DNA did not direct cleavage at
50.degree. C., either in the absence or presence of 50 mM KCl
(lanes 13 and 14). Thus, the specificity of the cleavage reactions
can be controlled by the extent of complementarity to the substrate
and by the conditions of incubation.
[0393] D. Cleavage of RNA
[0394] An shortened RNA version of the sequence used in the
transcleavage experiments discussed above was tested for its
ability to serve as a substrate in the reaction. The RNA is cleaved
at the expected place, in a reaction that is dependent upon the
presence of the pilot oligonucleotide. The RNA substrate, made by
T7 RNA polymerase in the presence of [.alpha.-.sup.32P]UTP,
corresponds to a truncated version of the DNA substrate used in
FIG. 12B. Reaction conditions were similar to those in used for the
DNA substrates described above, with 50 mM KCl; incubation was for
40 minutes at 55.degree. C. The pilot oligonucleotide used is
termed 30-0 (SEQ ID NO:20) and is shown in FIG. 13A.
[0395] The results of the cleavage reaction is shown in FIG. 13B.
The reaction was run either in the presence or absence of DNAPTaq
or pilot oligonucleotide as indicated in FIG. 13B.
[0396] Strikingly, in the case of RNA cleavage, a 3' arm is not
required for the pilot oligonucleotide. It is very unlikely that
this cleavage is due to previously described RNaseH, which would be
expected to cut the RNA in several places along the 30 base-pair
long RNA-DNA duplex. The 5' nuclease of DNAPTaq is a
structure-specific RNaseH that cleaves the RNA at a single site
near the 5' end of the heteroduplexed region.
[0397] It is surprising that an oligonucleotide lacking a 3' arm is
able to act as a pilot in directing efficient cleavage of an RNA
target because such oligonucleotides are unable to direct efficient
cleavage of DNA targets using native DNAPs. However, some 5'
nucleases of the present invention (for example, clones E, F and G
of FIG. 4) can cleave DNA in the absence of a 3' arm. In other
words, a non-extendable cleavage structure is not required for
specific cleavage with some 5' nucleases of the present invention
derived from thermostable DNA polymerases.
[0398] We tested whether cleavage of an RNA template by DNAPTaq in
the presence of a fully complementary primer could help explain why
DNAPTaq is unable to extend a DNA oligonucleotide on an RNA
template, in a reaction resembling that of reverse transcriptase.
Another thermophilic DNAP, DNAPTth, is able to use RNA as a
template, but only in the presence of Mn++, so we predicted that
this enzyme would not cleave RNA in the presence of this cation.
Accordingly, we incubated an RNA molecule with an appropriate pilot
oligonucleotide in the presence of DNAPTaq or DNAPTth, in buffer
containing either Mg++ or Mn++. As expected, both enzymes cleaved
the RNA in the presence of Mg++. However, DNAPTaq, but not DNAPTth,
degraded the RNA in the presence of Mn++. We conclude that the 5'
nuclease activities of many DNAPs may contribute to their inability
to use RNA as templates.
Example 2
Generation of 5' Nucleases from Thermostable DNA Polymerases
[0399] Thermostable DNA polymerases were generated which have
reduced synthetic activity, an activity that is an undesirable
side-reaction during DNA cleavage in the detection assay of the
invention, yet have maintained thermostable nuclease activity. The
result is a thermostable polymerase which cleaves nucleic acids DNA
with extreme specificity.
[0400] Type A DNA polymerases from eubacteria of the genus Thermus
share extensive protein sequence identity (90% in the
polymerization domain, using the Lipman-Pearson method in the DNA
analysis software from DNAStar, Wis.) and behave similarly in both
polymerization and nuclease assays. Therefore, we have used the
genes for the DNA polymerase of Thermus aquaticus (DNAPTaq) and
Thermus flavus (DNAPTf1) as representatives of this class.
Polymerase genes from other eubacterial organisms, such as Thermus
thernophilus, Thermus sp., Thermotoga maritima, Thermosipho
africanus and Bacillus stearothermophilus are equally suitable. The
DNA polymerases from these thermophilic organisms are capable of
surviving and performing at elevated temperatures, and can thus be
used in reactions in which temperature is used as a selection
against non-specific hybridization of nucleic acid strands.
[0401] The restriction sites used for deletion mutagenesis,
described below, were chosen for convenience. Different sites
situated with similar convenience are available in the Thermus
thermophilus gene and can be used to make similar constructs with
other Type A polymerase genes from related organisms.
[0402] A. Creation of 5' Nuclease Constructs
[0403] 1. Modified DNAPTaq Genes
[0404] The first step was to place a modified gene for the Taq DNA
polymerase on a plasmid under control of an inducible promoter. The
modified Taq polymerase gene was isolated as follows: The Taq DNA
polymerase gene was amplified by polymerase chain reaction from
genomic DNA from Thermus aquaticus, strain YT-1 (Lawyer et al.,
supra), using as primers the oligonucleotides described in SEQ ID
NOS:13-14. The resulting fragment of DNA has a recognition sequence
for the restriction endonuclease EcoRI at the 5' end of the coding
sequence and a BglII sequence at the 3' end. Cleavage with BglII
leaves a 5' overhang or "sticky end" that is compatible with the
end generated by BamHI. The PCR-amplified DNA was digested with
EcoRI and BamHI. The 2512 bp fragment containing the coding region
for the polymerase gene was gel purified and then ligated into a
plasmid which contains an inducible promoter.
[0405] In one embodiment of the invention, the pTTQ1 8 vector,
which contains the hybrid trp-lac (tac) promoter, was used [M. J.
R. Stark, Gene 5:255 (1987)] and shown in FIG. 14. The tac promoter
is under the control of the E. coli lac repressor. Repression
allows the synthesis of the gene product to be suppressed until the
desired level of bacterial growth has been achieved, at which point
repression is removed by addition of a specific inducer,
isopropyl-.beta.-D-thiogalactopyranoside (IPTG). Such a system
allows the expression of foreign proteins that may slow or prevent
growth of transformants.
[0406] Bacterial promoters, such as tac, may not be adequately
suppressed when they are present on a multiple copy plasmid. If a
highly toxic protein is placed under control of such a promoter,
the small amount of expression leaking through can be harmful to
the bacteria. In another embodiment of the invention, another
option for repressing synthesis of a cloned gene product was used.
The non-bacterial promoter, from bacteriophage T7, found in the
plasmid vector series pET-3 was used to express the cloned mutant
Taq polymerase genes [FIG. 15; Studier and Moffatt, J. Mol. Biol.
189:113 (1986)]. This promoter initiates transcription only by T7
RNA polymerase. In a suitable strain, such as BL21(DE3)pLYS, the
gene for this RNA polymerase is carried on the bacterial genome
under control of the lac operator. This arrangement has the
advantage that expression of the multiple copy gene (on the
plasmid) is completely dependent on the expression of T7 RNA
polymerase, which is easily suppressed because it is present in a
single copy.
[0407] For ligation into the pTTQ18 vector (FIG. 14), the PCR
product DNA containing the Taq polymerase coding region (mutTaq,
clone 4B, SEQ ID NO:21) was digested with EcoRI and BglII and this
fragment was ligated under standard "sticky end" conditions
[Sambrook et al. Molecular Cloning, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, pp. 1.63-1.69 (1989)] into the EcoRI and
BamHI sites of the plasmid vector pTTQ18. Expression of this
construct yields a translational fusion product in which the first
two residues of the native protein (Met-Arg) are replaced by three
from the vector (Met-Asn-Ser), but the remainder of the natural
protein would not change. The construct was transformed into the
JM109 strain of E. coli and the transformants were plated under
incompletely repressing conditions that do not permit growth of
bacteria expressing the native protein. These plating conditions
allow the isolation of genes containing pre-existing mutations,
such as those that result from the infidelity of Taq polymerase
during the amplification process.
[0408] Using this amplification/selection protocol, we isolated a
clone (depicted in FIG. 4B) containing a mutated Taq polymerase
gene (mutTaq, clone 4B). The mutant was first detected by its
phenotype, in which temperature-stable 5' nuclease activity in a
crude cell extract was normal, but polymerization activity was
almost absent (approximately less than 1% of wild type Taq
polymerase activity).
[0409] DNA sequence analysis of the recombinant gene showed that it
had changes in the polymerase domain resulting in two amino acid
substitutions: an A to G change at nucleotide position 1394 causes
a Glu to Gly change at amino acid position 465 (numbered according
to the natural nucleic and amino acid sequences, SEQ ID NOS:1 and
4) and another A to G change at nucleotide position 2260 causes a
Gln to Arg change at amino acid position 754. Because the Gln to
Gly mutation is at a nonconserved position and because the Glu to
Arg mutation alters an amino acid that is conserved in virtually
all of the known Type A polymerases, this latter mutation is most
likely the one responsible for curtailing the synthesis activity of
this protein. The nucleotide sequence for the FIG. 4B construct is
given in SEQ ID NO:21. The enzyme encoded by this sequence is
referred to as Cleavase.RTM. A/G.
[0410] Subsequent derivatives of DNAPTaq constructs were made from
the mutTaq gene, thus, they all bear these amino acid substitutions
in addition to their other alterations, unless these particular
regions were deleted. These mutated sites are indicated by black
boxes at these locations in the diagrams in FIG. 4. In FIG. 4, the
designation "3' Exo" is used to indicate the location of the 3'
exonuclease activity associated with Type A polymerases which is
not present in DNAPTaq. All constructs except the genes shown in
FIGS. 4E, F and G were made in the pTTQ 18 vector.
[0411] The cloning vector used for the genes in FIGS. 4E and F was
from the commercially available pET-3 series, described above.
Though this vector series has only a BamHI site for cloning
downstream of the T7 promoter, the series contains variants that
allow cloning into any of the three reading frames. For cloning of
the PCR product described above, the variant called pET-3c was used
(FIG. 15). The vector was digested with BamHI, dephosphorylated
with calf intestinal phosphatase, and the sticky ends were filled
in using the Klenow fragment of DNAPEc1 and dNTPs. The gene for the
mutant Taq DNAP shown in FIG. 4B (mutTaq, clone 4B) was released
from pTTQ18 by digestion with EcoRI and SalI, and the "sticky ends"
were filled in as was done with the vector. The fragment was
ligated to the vector under standard blunt-end conditions (Sambrook
et al., Molecular Cloning, supra), the construct was transformed
into the BL21(DE3)pLYS strain of E. coli, and isolates were
screened to identify those that were ligated with the gene in the
proper orientation relative to the promoter. This construction
yields another translational fusion product, in which the first two
amino acids of DNAPTaq (Met-Arg) are replaced by 13 from the vector
plus two from the PCR primer
(Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Ile-Asn-Ser) (SEQ
ID NO:29).
[0412] Our goal was to generate enzymes that lacked the ability to
synthesize DNA, but retained the ability to cleave nucleic acids
with a 5' nuclease activity. The act of primed, templated synthesis
of DNA is actually a coordinated series of events, so it is
possible to disable DNA synthesis by disrupting one event while not
affecting the others. These steps include, but are not limited to,
primer recognition and binding, dNTP binding and catalysis of the
inter-nucleotide phosphodiester bond. Some of the amino acids in
the polymerization domain of DNAPEcI have been linked to these
functions, but the precise mechanisms are as yet poorly
defined.
[0413] One way of destroying the polymerizing ability of a DNA
polymerase is to delete all or part of the gene segment that
encodes that domain for the protein, or to otherwise render the
gene incapable of making a complete polymerization domain.
Individual mutant enzymes may differ from each other in stability
and solubility both inside and outside cells. For instance, in
contrast to the 5' nuclease domain of DNAPEcI, which can be
released in an active form from the polymerization domain by gentle
proteolysis [Setlow and Kornberg, J. Biol. Chem. 247:232 (1972)],
the Thermus nuclease domain, when treated similarly, becomes less
soluble and the cleavage activity is often lost.
[0414] Using the mutant gene shown in FIG. 4B as starting material,
several deletion constructs were created. All cloning technologies
were standard (Sambrook et al., supra) and are summarized briefly,
as follows:
[0415] FIG. 4C: The mutTaq construct was digested with PstI, which
cuts once within the polymerase coding region, as indicated, and
cuts immediately downstream of the gene in the multiple cloning
site of the vector. After release of the fragment between these two
sites, the vector was re-ligated, creating an 894-nucleotide
deletion, and bringing into frame a stop codon 40 nucleotides
downstream of the junction. The nucleotide sequence of this 5'
nuclease (clone 4C) is given in SEQ ID NO:9.
[0416] FIG. 4D: The mutTaq construct was digested with NheI, which
cuts once in the gene at position 2047. The resulting
four-nucleotide 5' overhanging ends were filled in, as described
above, and the blunt ends were re-ligated. The resulting
four-nucleotide insertion changes the reading frame and causes
termination of translation ten amino acids downstream of the
mutation. The nucleotide sequence of this 5' nuclease (clone 4D) is
given in SEQ ID NO: 10.
[0417] FIG. 4E: The entire mutTaq gene was cut from pTTQ18 using
EcoRI and SalI and cloned into pET-3c, as described above. This
clone was digested with BstXI and XcmI, at unique sites that are
situated as shown in FIG. 4E. The DNA was treated with the Klenow
fragment of DNAPEc1 and dNTPs, which resulted in the 3' overhangs
of both sites being trimmed to blunt ends. These blunt ends were
ligated together, resulting in an out-of-frame deletion of 1540
nucleotides. An in-frame termination codon occurs 18 triplets past
the junction site. The nucleotide sequence of this 5' nuclease
(clone 4E) is given in SEQ ID NO: 11, with the appropriate leader
sequence given in SEQ ID NO:30. It is also referred to as
Cleavase.RTM. BX.
[0418] FIG. 4F: The entire mutTaq gene was cut from pTTQ18 using
EcoRI and SalI and cloned into pET-3c, as described above. This
clone was digested with BstXI and BamHI, at unique sites that are
situated as shown in the diagram. The DNA was treated with the
Klenow fragment of DNAPEc1 and dNTPs, which resulted in the 3'
overhang of the BstXI site being trimmed to a blunt end, while the
5' overhang of the BamHI site was filled in to make a blunt end.
These ends were ligated together, resulting in an in-frame deletion
of 903 nucleotides. The nucleotide sequence of the 5' nuclease
(clone 4F) is given in SEQ ID NO:12. It is also referred to as
Cleavase.RTM. BB.
[0419] FIG. 4G: This polymerase is a variant of that shown in FIG.
4E. It was cloned in the plasmid vector pET-21 (Novagen). The
non-bacterial promoter from bacteriophage T7, found in this vector,
initiates transcription only by T7 RNA polymerase. See Studier and
Moffatt, supra. In a suitable strain, such as (DES)pLYS, the gene
for this RNA polymerase is carried on the bacterial genome under
control of the lac operator. This arrangement has the advantage
that expression of the multiple copy gene (on the plasmid) is
completely dependent on the expression of T7 RNA polymerase, which
is easily suppressed because it is present in a single copy.
Because the expression of these mutant genes is under this tightly
controlled promoter, potential problems of toxicity of the
expressed proteins to the host cells are less of a concern.
[0420] The pET-21 vector also features a "His*Tag", a stretch of
six consecutive histidine residues that are added on the carboxy
terminus of the expressed proteins. The resulting proteins can then
be purified in a single step by metal chelation chromatography,
using a commerically available (Novagen) column resin with
immobilized Ni.sup.++ ions. The 2.5 ml columns are reusable, and
can bind up to 20 mg of the target protein under native or
denaturing (guanidine*HCl or urea) conditions.
[0421] E. coli (DES)pLYS cells are transformed with the constructs
described above using standard transformation techniques, and used
to inoculate a standard growth medium (e.g., Luria-Bertani broth).
Production of T7 RNA polymerase is induced during log phase growth
by addition of IPTG and incubated for a further 12 to 17 hours.
Aliquots of culture are removed both before and after induction and
the proteins are examined by SDS-PAGE. Staining with Coomassie Blue
allows visualization of the foreign proteins if they account for
about 3-5% of the cellular protein and do not co-migrate with any
of the major protein bands. Proteins that co-migrate with major
host protein must be expressed as more than 10% of the total
protein to be seen at this tage of analysis.
[0422] Some mutant proteins are sequestered by the cells into
inclusion bodies. These are granules that form in the cytoplasm
when bacteria are made to express high levels of a foreign protein,
and they can be purified from a crude lysate, and analyzed by
SDS-PAGE to determine their protein content. If the cloned protein
is found in the inclusion bodies, it must be released to assay the
cleavage and polymerase activities. Different methods of
solubilization may be appropriate for different proteins, and a
variety of methods are known. See e.g., Builder & Ogez, U.S.
Pat. No. 4,511,502 (1985); Olson, U.S. Pat. No. 4,518,526 (1985);
Olson & Pai, U.S. Pat. No. 4,511,503 (1985); Jones et al., U.S.
Pat. No. 4,512,922 (1985), all of which are hereby incorporated by
reference.
[0423] The solubilized protein is then purified on the Ni.sup.++
column as described above, following the manufacturers instructions
(Novagen). The washed proteins are eluted from the column by a
combination of imidazole competitor (1 M) and high salt (0.5 M
NaCl), and dialyzed to exchange the buffer and to allow denature
proteins to refold. Typical recoveries result in approximately 20
.mu.g of specific protein per ml of starting culture. The DNAP
mutant is referred to as the Cleavase.RTM. BN nuclease and the
sequence is given in SEQ ID NO:31 (the amino acid sequence of the
Cleavase.RTM. BN nuclease is obtained by translating the DNA
sequence of SEQ ID NO:31).
[0424] 2. Modified DNAPTf1 Gene
[0425] The DNA polymerase gene of Thermus flavus was isolated from
the "T. flavus" AT-62 strain obtained from the American Type Tissue
Collection (ATCC 33923). This strain has a different restriction
map then does the T. flavus strain used to generate the sequence
published by Akhmetzjanov and Vakhitov, supra. The published
sequence is listed as SEQ ID NO:2. No sequence data has been
published for the DNA polymerase gene from the AT-62 strain of T.
flavus.
[0426] Genomic DNA from T. flavus was amplified using the same
primers used to amplify the T. aquaticus DNA polymerase gene (SEQ
ID NOS:13-14). The approximately 2500 base pair PCR fragment was
digested with EcoRI and BamHI. The over-hanging ends were made
blunt with the Klenow fragment of DNAPEc1 and dNTPs. The resulting
approximately 1800 base pair fragment containing the coding region
for the N-terminus was ligated into pET-3c, as described above.
This construct, clone 5B, is depicted in FIG. 5B. The wild type T.
flavus DNA polymerase gene is depicted in FIG. 5A. The 5B clone has
the same leader amino acids as do the DNAPTaq clones 4E and F which
were cloned into pET-3c; it is not known precisely where
translation termination occurs, but the vector has a strong
transcription termination signal immediately downstream of the
cloning site.
[0427] B. Growth and Induction of Transformed Cells
[0428] Bacterial cells were transformed with the constructs
described above using standard transformation techniques and used
to inoculate 2 mls of a standard growth medium (e.g., Luria-Bertani
broth). The resulting cultures were incubated as appropriate for
the particular strain used, and induced if required for a
particular expression system. For all of the constructs depicted in
FIGS. 4 and 5, the cultures were grown to an optical density (at
600 nm wavelength) of 0.5 OD.
[0429] To induce expression of the cloned genes, the cultures were
brought to a final concentration of 0.4 mM IPTG and the incubations
were continued for 12 to 17 hours. 50 .mu.l aliquots of each
culture were removed both before and after induction and were
combined with 20 .mu.l of a standard gel loading buffer for sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Subsequent staining with Coomassie Blue (Sambrook et al., supra)
allows visualization of the foreign proteins if they account for
about 3-5% of the cellular protein and do not co-migrate with any
of the major E. coli protein bands. Proteins that do co-migrate
with a major host protein must be expressed as more than 10% of the
total protein to be seen at this stage of analysis.
[0430] C. Heat Lysis and Fractionation
[0431] Expressed thermostable proteins, i.e., the 5' nucleases,
were isolated by heating crude bacterial cell extracts to cause
denaturation and precipitation of the less stable E. coli proteins.
The precipitated E. coli proteins were then, along with other cell
debris, removed by centrifugation. 1.7 mls of the culture were
pelleted by microcentrifugation at 12,000 to 14,000 rpm for 30 to
60 seconds. After removal of the supernatant, the cells were
resuspended in 400 .mu.l of buffer A (50 mM Tris-HCl, pH 7.9, 50 mM
dextrose, 1 mM EDTA), re-centrifuged, then resuspended in 80 .mu.l
of buffer A with 4 mg/ml lysozyme. The cells were incubated at room
temperature for 15 minutes, then combined with 80 .mu.l of buffer B
(10 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM PMSF, 0.5%
Tween-20, 0.5% Nonidet-P40).
[0432] This mixture was incubated at 75.degree. C. for 1 hour to
denature and precipitate the host proteins. This cell extract was
centrifuged at 14,000 rpm for 15 minutes at 4.degree. C., and the
supernatant was transferred to a fresh tube. An aliquot of 0.5 to 1
.mu.l of this supernatant was used directly in each test reaction,
and the protein content of the extract was determined by subjecting
7 .mu.l to electrophoretic analysis, as above. The native
recombinant Taq DNA polymerase [Englke, Anal. Biochem 191:396
(1990)], and the double point mutation protein shown in FIG. 4B are
both soluble and active at this point.
[0433] The foreign protein may not be detected after the heat
treatments due to sequestration of the foreign protein by the cells
into inclusion bodies. These are granules that form in the
cytoplasm when bacteria are made to express high levels of a
foreign protein, and they can be purified from a crude lysate, and
analyzed SDS PAGE to determine their protein content. Many methods
have been described in the literature, and one approach is
described below.
[0434] D. Isolation and Solubilization of Inclusion Bodies
[0435] A small culture was grown and induced as described above. A
1.7 ml aliquot was pelleted by brief centrifugation, and the
bacterial cells were resuspended in 100 .mu.l of Lysis buffer (50
mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl). 2.5 .mu.l of 20 mM
PMSF were added for a final concentration of 0.5 mM, and lysozyme
was added to a concentration of 1.0 mg/ml. The cells were incubated
at room temperature for 20 minutes, deoxycholic acid was added to 1
mg/ml (1 .mu.l of 100 mg/ml solution), and the mixture was further
incubated at 37.degree. C. for about 15 minutes or until viscous.
DNAse I was added to 10 .mu.g/ml and the mixture was incubated at
room temperature for about 30 minutes or until it was no longer
viscous.
[0436] From this mixture the inclusion bodies were collected by
centrifugation at 14,000 rpm for 15 minutes at 4.degree. C., and
the supernatant was discarded. The pellet was resuspended in 100
.mu.l of lysis buffer with 10 mM EDTA (pH 8.0) and 0.5% Triton
X-100. After 5 minutes at room temperature, the inclusion bodies
were pelleted as before, and the supernatant was saved for later
analysis. The inclusion bodies were resuspended in 50 .mu.l of
distilled water, and 5 .mu.l was combined with SDS gel loading
buffer (which dissolves the inclusion bodies) and analyzed
electrophoretically, along with an aliquot of the supernatant.
[0437] If the cloned protein is found in the inclusion bodies, it
may be released to assay the cleavage and polymerase activities and
the method of solubilization must be compatible with the particular
activity. Different methods of solubilization may be appropriate
for different proteins, and a variety of methods are discussed in
Molecular Cloning (Sambrook et al., supra). The following is an
adaptation we have used for several of our isolates.
[0438] 20 .mu.l of the inclusion body-water suspension were
pelleted by centrifugation at 14,000 rpm for 4 minutes at room
temperature, and the supernatant was discarded. To further wash the
inclusion bodies, the pellet was resuspended in .sup.20 .mu.l of
lysis buffer with 2M urea, and incubated at room temperature for
one hour. The washed inclusion bodies were then resuspended in 2
.mu.l of lysis buffer with 8M urea; the solution clarified visibly
as the inclusion bodies dissolved. Undissolved debris was removed
by centrifugation at 14,000 rpm for 4 minutes at room temperature,
and the extract supernatant was transferred to a fresh tube.
[0439] To reduce the urea concentration, the extract was diluted
into KH.sub.2PO.sub.4. A fresh tube was prepared containing 180
.mu.l of 50 mM KH.sub.2PO.sub.4, pH 9.5, 1 mM EDTA and 50 mM NaCl.
A 2 A.sup.1 aliquot of the extract was added and vortexed briefly
to mix. This step was repeated until all of the extract had been
added for a total of 10 additions. The mixture was allowed to sit
at room temperature for 15 minutes, during which time some
precipitate often forms. Precipitates were removed by
centrifugation at 14,000 rpm, for 15 minutes at room temperature,
and the supernatant was transferred to a fresh tube. To the 200
.mu.l of protein in the KH.sub.2PO.sub.4 solution, 140-200 .mu.l of
saturated (NH.sub.4).sub.2SO.sub.4 were added, so that the
resulting mixture was about 41% to 50% saturated
(NH.sub.4).sub.2SO.sub.4. The mixture was chilled on ice for 30
minutes to allow the protein to precipitate, and the protein was
then collected by centrifugation at 14,000 rpm, for 4 minutes at
room temperature. The supernatant was discarded, and the pellet was
dissolved in 20 A.sup.1 Buffer C (20 mM HEPES, pH 7.9, 1 mM EDTA,
0.5% PMSF, 25 mM KCl and 0.5% each of Tween-20 and Nonidet P 40).
The protein solution was centrifuged again for 4 minutes to pellet
insoluble materials, and the supernatant was removed to a fresh
tube. The protein contents of extracts prepared in this manner were
visualized by resolving 1-4 .mu.l by SDS-PAGE; 0.5 to 1 .mu.l of
extract was tested in the cleavage and polymerization assays as
described.
[0440] E. Protein Analysis for Presence of Nuclease and Synthetic
Activity
[0441] The 5' nucleases described above and shown in FIGS. 4 and 5
were analyzed by the following methods.
[0442] 1. Structure Specific Nuclease Assay
[0443] A candidate modified polymerase is tested for 5' nuclease
activity by examining its ability to catalyze structure-specific
cleavages. By the term "cleavage structure" as used herein, is
meant a nucleic acid structure which is a substrate for cleavage by
the 5' nuclease activity of a DNAP.
[0444] The polymerase is exposed to test complexes that have the
structures shown in FIG. 16. Testing for 5' nuclease activity
involves three reactions: 1) a primer-directed cleavage (FIG. 16B)
is performed because it is relatively insensitive to variations in
the salt concentration of the reaction and can, therefore, be
performed in whatever solute conditions the modified enzyme
requires for activity; this is generally the same conditions
preferred by unmodified polymerases; 2) a similar primer-directed
cleavage is performed in a buffer which permits primer-independent
cleavage, i.e., a low salt buffer, to demonstrate that the enzyme
is viable under these conditions; and 3) a primer-independent
cleavage (FIG. 16A) is performed in the same low salt buffer.
[0445] The bifurcated duplex is formed between a substrate strand
and a template strand as shown in FIG. 16. By the term "substrate
strand" as used herein, is meant that strand of nucleic acid in
which the cleavage mediated by the 5' nuclease activity occurs. The
substrate strand is always depicted as the top strand in the
bifurcated complex which serves as a substrate for 5' nuclease
cleavage (FIG. 16). By the term "template strand" as used herein,
is meant the strand of nucleic acid which is at least partially
complementary to the substrate strand and which anneals to the
substrate strand to form the cleavage structure. The template
strand is always depicted as the bottom strand of the bifurcated
cleavage structure (FIG. 16). If a primer (a short oligonucleotide
of 19 to 30 nucleotides in length) is added to the complex, as when
primer-dependent cleavage is to be tested, it is designed to anneal
to the 3' arm of the template strand (FIG. 16B). Such a primer
would be extended along the template strand if the polymerase used
in the reaction has synthetic activity.
[0446] The cleavage structure may be made as a single hairpin
molecule, with the 3' end of the target and the 5' end of the pilot
joined as a loop as shown in FIG. 16E. A primer oligonucleotide
complementary to the 3' arm is also required for these tests so
that the enzyme's sensitivity to the presence of a primer may be
tested.
[0447] Nucleic acids to be used to form test cleavage structures
can be chemically synthesized, or can be generated by standard
recombinant DNA techniques. By the latter method, the hairpin
portion of the molecule can be created by inserting into a cloning
vector duplicate copies of a short DNA segment, adjacent to each
other but in opposing orientation. The double-stranded fragment
encompassing this inverted repeat, and including enough flanking
sequence to give short (about 20 nucleotides) unpaired 5' and 3'
arms, can then be released from the vector by restriction enzyme
digestion, or by PCR performed with an enzyme lacking a 5'
exonuclease (e.g., the Stoffel fragment of Amplitaq.TM. DNA
polymerase, Vent.TM. DNA polymerase).
[0448] The test DNA can be labeled on either end, or internally,
with either a radioisotope, or with a non-isotopic tag. Whether the
hairpin DNA is a synthetic single strand or a cloned double strand,
the DNA is heated prior to use to melt all duplexes. When cooled on
ice, the structure depicted in FIG. 16E is formed, and is stable
for sufficient time to perform these assays.
[0449] To test for primer-directed cleavage (Reaction 1), a
detectable quantity of the test molecule (typically 1-100 fmol of
.sup.32P-labeled hairpin molecule) and a 10 to 100-fold molar
excess of primer are placed in a buffer known to be compatible with
the test enzyme. For Reaction 2, where primer-directed cleavage is
performed under condition which allow primer-independent cleavage,
the same quantities of molecules are placed in a solution that is
the same as the buffer used in Reaction 1 regarding pH, enzyme
stabilizers (e.g., bovine serum albumin, nonionic detergents,
gelatin) and reducing agents (e.g., dithiothreitol,
2-mercaptoethanol) but that replaces any monovalent cation salt
with 20 mM KCl; 20 mM KCl is the demonstrated optimum for
primer-independent cleavage. Buffers for enzymes, such as DNAPEc1,
that usually operate in the absence of salt are not supplemented to
achieve this concentration. To test for primer-independent cleavage
(Reaction 3) the same quantity of the test molecule, but no primer,
are combined under the same buffer conditions used for Reaction
2.
[0450] All three test reactions are then exposed to enough of the
enzyme that the molar ratio of enzyme to test complex is
approximately 1:1. The reactions are incubated at a range of
temperatures up to, but not exceeding, the temperature allowed by
either the enzyme stability or the complex stability, whichever is
lower, up to 80.degree. C. for enzymes from thermophiles, for a
time sufficient to allow cleavage (10 to 60 minutes). The products
of Reactions 1, 2 and 3 are resolved by denaturing polyacrylamide
gel electrophoresis, and visualized by autoradiography or by a
comparable method appropriate to the labeling system used.
Additional labeling systems include chemiluminescence detection,
silver or other stains, blotting and probing and the like. The
presence of cleavage products is indicated by the presence of
molecules which migrate at a lower molecular weight than does the
uncleaved test structure. These cleavage products indicate that the
candidate polymerase has structure-specific 5' nuclease
activity.
[0451] To determine whether a modified DNA polymerase has
substantially the same 5' nuclease activity as that of the native
DNA polymerase, the results of the above-described tests are
compared with the results obtained from these tests performed with
the native DNA polymerase. By "substantially the same 5"nuclease
activity" we mean that the modified polymerase and the native
polymerase will both cleave test molecules in the same manner. It
is not necessary that the modified polymerase cleave at the same
rate as the native DNA polymerase.
[0452] Some enzymes or enzyme preparations may have other
associated or contaminating activities that may be functional under
the cleavage conditions described above and that may interfere with
5' nuclease detection. Reaction conditions can be modified in
consideration of these other activities, to avoid destruction of
the substrate, or other masking of the 5' nuclease cleavage and its
products. For example, the DNA polymerase I of E. coli (Pol I), in
addition to its polymerase and 5' nuclease activities, has a 3'
exonuclease that can degrade DNA in a 3' to 5' direction.
Consequently, when the molecule in FIG. 16E is exposed to this
polymerase under the conditions described above, the 3' exonuclease
quickly removes the unpaired 3' arm, destroying the bifurcated
structure required of a substrate for the 5' exonuclease cleavage
and no cleavage is detected. The true ability of Pol I to cleave
the structure can be revealed if the 3' exonuclease is inhibited by
a change of conditions (e.g. pH), mutation, or by addition of a
competitor for the activity. Addition of 500 pmoles of a
single-stranded competitor oligonucleotide, unrelated to the FIG.
16E structure, to the cleavage reaction with Pol I effectively
inhibits the digestion of the 3' arm of the FIG. 16E structure
without interfering with the 5' exonuclease release of the 5' arm.
The concentration of the competitor is not critical, but should be
high enough to occupy the 3' exonuclease for the duration of the
reaction.
[0453] Similar destruction of the test molecule may be caused by
contaminants in the candidate polymerase preparation. Several sets
of the structure specific nuclease reactions may be performed to
determine the purity of the candidate nuclease and to find the
window between under and over exposure of the test molecule to the
polymerase preparation being investigated.
[0454] The above described modified polymerases were tested for 5'
nuclease activity as follows: Reaction 1 was performed in a buffer
of 10 mM Tris-Cl, pH 8.5 at 20.degree. C., 1.5 mM MgCl.sub.2 and 50
mM KCl and in Reaction 2 the KCl concentration was reduced to 20
mM. In Reactions 1 and 2, 10 fmoles of the test substrate molecule
shown in FIG. 16E were combined with 1 pmole of the indicated
primer and 0.5 to 1.0 .mu.l of extract containing the modified
polymerase (prepared as described above). This mixture was then
incubated for 10 minutes at 55.degree. C. For all of the mutant
polymerases tested these conditions were sufficient to give
complete cleavage. When the molecule shown in FIG. 16E was labeled
at the 5' end, the released 5' fragment, 25 nucleotides long, was
conveniently resolved on a 20% polyacrylamide gel (19:1
cross-linked) with 7 M urea in a buffer containing 45 mM
Tris-borate pH 8.3, 1.4 mM EDTA. Clones 4C-F and 5B exhibited
structure-specific cleavage comparable to that of the unmodified
DNA polymerase. Additionally, clones 4E, 4F and 4G have the added
ability to cleave DNA in the absence of a 3' arm as discussed
above. Representative cleavage reactions are shown in FIG. 17.
[0455] For the reactions shown in FIG. 17, the mutant polymerase
clones 4E (Taq mutant) and 5B (Tfl mutant) were examined for their
ability to cleave the hairpin substrate molecule shown in FIG. 16E.
The substrate molecule was labeled at the 5' terminus with
.sup.32P. 10 fmoles of heat-denatured, end-labeled substrate DNA
and 0.5 units of DNAPTaq (lane 1) or 0.5 .mu.l of 4e or 5b extract
(FIG. 17, lanes 2-7, extract was prepared as described above) were
mixed together in a buffer containing 10 mM Tris-Cl, pH 8.5, 50 mM
KCl and 1.5 mM MgCl.sub.2. The final reaction volume was 10 .mu.l.
Reactions shown in lanes 4 and 7 contain in addition 50 .mu.M of
each dNTP. Reactions shown in lanes 3, 4, 6 and 7 contain 0.2 .mu.M
of the primer oligonucleotide (complementary to the 3' arm of the
substrate and shown in FIG. 16E). Reactions were incubated at
55.degree. C. for 4 minutes. Reactions were stopped by the addition
of 8 .mu.l of 95% formamide containing 20 mM EDTA and 0.05% marker
dyes per 10 .mu.l reaction volume. Samples were then applied to 12%
denaturing acrylamide gels. Following electrophoresis, the gels
were autoradiographed. FIG. 17 shows that clones 4E and 5B exhibit
cleavage activity similar to that of the native DNAPTaq. Note that
some cleavage occurs in these reactions in the absence of the
primer. When long hairpin structure, such as the one used here
(FIG. 16E), are used in cleavage reactions performed in buffers
containing 50 mM KCl a low level of primer-independent cleavage is
seen. Higher concentrations of KCl suppress, but do not eliminate,
this primer-independent cleavage under these conditions.
[0456] 2. Assay for Synthetic Activity
[0457] The ability of the modified enzyme or proteolytic fragments
is assayed by adding the modified enzyme to an assay system in
which a primer is annealed to a template and DNA synthesis is
catalyzed by the added enzyme. Many standard laboratory techniques
employ such an assay. For example, nick translation and enzymatic
sequencing involve extension of a primer along a DNA template by a
polymerase molecule.
[0458] In a preferred assay for determining the synthetic activity
of a modified enzyme an oligonucleotide primer is annealed to a
single-stranded DNA template, e.g., bacteriophage M13 DNA, and the
primer/template duplex is incubated in the presence of the modified
polymerase in question, deoxynucleoside triphosphates (dNTPs) and
the buffer and salts known to be appropriate for the unmodified or
native enzyme. Detection of either primer extension (by denaturing
gel electrophoresis) or dNTP incorporation (by acid precipitation
or chromatography) is indicative of an active polymerase. A label,
either isotopic or non-isotopic, is preferably included on either
the primer or as a dNTP to facilitate detection of polymerization
products. Synthetic activity is quantified as the amount of free
nucleotide incorporated into the growing DNA chain and is expressed
as amount incorporated per unit of time under specific reaction
conditions.
[0459] Representative results of an assay for synthetic activity is
shown in FIG. 18. The synthetic activity of the mutant DNAPTaq
clones 4B-F was tested as follows: A master mixture of the
following buffer was made: 1.2.times.PCR buffer (1.times.PCR buffer
contains 50 mM KCl, 1.5 mM MgCl.sub.2, 10 mM Tris-Cl, ph 8.5 and
0.05% each Tween 20 and Nonidet P40), 50 .mu.M each of dGTP, dATP
and dTTP, 5 .mu.M dCTP and 0.125 .mu.M .alpha.-.sup.32P-dCTP at 600
Ci/mmol. Before adjusting this mixture to its final volume, it was
divided into two equal aliquots. One received distilled water up to
a volume of 50 .mu.l to give the concentrations above. The other
received 5 .mu.g of single-stranded M13 mp18 DNA (approximately 2.5
pmol or 0.05 .mu.M final concentration) and 250 pmol of M13
sequencing primer (5 .mu.M final concentration) and distilled water
to a final volume of 50 .mu.l. Each cocktail was warmed to
75.degree. C. for 5 minutes and then cooled to room temperature.
This allowed the primers to anneal to the DNA in the DNA-containing
mixtures.
[0460] For each assay, 4 .mu.l of the cocktail with the DNA was
combined with 1 .mu.l of the mutant polymerase, prepared as
described, or 1 unit of DNAPTaq (Perkin Elmer) in 1 .mu.l of
dH.sub.2O. A "no DNA" control was done in the presence of the
DNAPTaq (FIG. 18, lane 1), and a "no enzyme" control was done using
water in place of the enzyme (lane 2). Each reaction was mixed,
then incubated at room temperature (approx. 22.degree. C.) for 5
minutes, then at 55.degree. C. for 2 minutes, then at 72.degree. C.
for 2 minutes. This step incubation was done to detect
polymerization in any mutants that might have optimal temperatures
lower than 72.degree. C. After the final incubation, the tubes were
spun briefly to collect any condensation and were placed on ice.
One .mu.l of each reaction was spotted at an origin 1.5 cm from the
bottom edge of a polyethyleneimine (PEI) cellulose thin layer
chromatography plate and allowed to dry. The chromatography plate
was run in 0.75 M NaH.sub.2PO.sub.4, pH 3.5, until the buffer front
had run approximately 9 cm from the origin. The plate was dried,
wrapped in plastic wrap, marked with luminescent ink, and exposed
to X-ray film. Incorporation was detected as counts that stuck
where originally spotted, while the unincorporated nucleotides were
carried by the salt solution from the origin.
[0461] Comparison of the locations of the counts with the two
control lanes confirmed the lack of polymerization activity in the
mutant preparations. Among the modified DNAPTaq clones, only clone
4B retains any residual synthetic activity as shown in FIG. 18.
Example 3
5' Nucleases Derived from Thermostable DNA Polymerases can Cleave
Short Hairpin Structures with Specificity
[0462] The ability of the 5' nucleases to cleave hairpin structures
to generate a cleaved hairpin structure suitable as a detection
molecule was examined. The structure and sequence of the hairpin
test molecule is shown in FIG. 19A (SEQ ID NO:15). The
oligonucleotide (labeled "primer" in FIG. 19A, SEQ ID NO:22) is
shown annealed to its complementary sequence on the 3' arm of the
hairpin test molecule. The hairpin test molecule was single-end
labeled with .sup.32P using a labeled T7 promoter primer in a
polymerase chain reaction. The label is present on the 5' arm of
the hairpin test molecule and is represented by the star in FIG.
19A.
[0463] The cleavage reaction was performed by adding 10 fmoles of
heat-denatured, end-labeled hairpin test molecule, 0.2 uM of the
primer oligonucleotide (complementary to the 3' arm of the
hairpin), 50 .mu.M of each dNTP and 0.5 units of DNAPTaq (Perkin
Elmer) or 0.5 .mu.l of extract containing a 5' nuclease (prepared
as described above) in a total volume of 10 .mu.l in a buffer
containing 10 mM Tris-Cl, pH 8.5, 50 mM KCl and 1.5 mM MgCl.sub.2.
Reactions shown in lanes 3, 5 and 7 were run in the absence of
dNTPs.
[0464] Reactions were incubated at 55.degree. C. for 4 minutes.
Reactions were stopped at 55.degree. C. by the addition of 8 .mu.l
of 95% formamide with 20 mM EDTA and 0.05% marker dyes per 10 .mu.l
reaction volume. Samples were not heated before loading onto
denaturing polyacrylamide gels (10% polyacrylamide, 19:1
crosslinking, 7 M urea, 89 mM Tris-borate, pH 8.3, 2.8 mM EDTA).
The samples were not heated to allow for the resolution of
single-stranded and re-duplexed uncleaved hairpin molecules.
[0465] FIG. 19B shows that altered polymerases lacking any
detectable synthetic activity cleave a hairpin structure when an
oligonucleotide is annealed to the single-stranded 3' arm of the
hairpin to yield a single species of cleaved product (FIG. 19B,
lanes 3 and 4). 5' nucleases, such as clone 4D, shown in lanes 3
and 4, produce a single cleaved product even in the presence of
dNTPs. 5' nucleases which retain a residual amount of synthetic
activity (less than 1% of wild type activity) produce multiple
cleavage products as the polymerase can extend the oligonucleotide
annealed to the 3' arm of the hairpin thereby moving the site of
cleavage (clone 4B, lanes 5 and 6). Native DNATaq produces even
more species of cleavage products than do mutant polymerases
retaining residual synthetic activity and additionally converts the
hairpin structure to a double-stranded form in the presence of
dNTPs due to the high level of synthetic activity in the native
polymerase (FIG. 19B, lane 8).
Example 4
[0466] Test of the Trigger/Detection Assay
[0467] To test the ability of an oligonucleotide of the type
released in the trigger reaction of the trigger/detection assay to
be detected in the detection reaction of the assay, the two hairpin
structures shown in FIG. 20A were synthesized using standard
techniques. The two hairpins are termed the A-hairpin (SEQ ID
NO:23) and the T-hairpin (SEQ ID NO:24). The predicted sites of
cleavage in the presence of the appropriate annealed primers are
indicated by the arrows. The A- and T-hairpins were designed to
prevent intra-strand mis-folding by omitting most of the T residues
in the A-hairpin and omitting most of the A residues in the
T-hairpin. To avoid mis-priming and slippage, the hairpins were
designed with local variations in the sequence motifs (e.g.,
spacing T residues one or two nucleotides apart or in pairs). The
A- and T-hairpins can be annealed together to form a duplex which
has appropriate ends for directional cloning in pUC-type vectors;
restriction sites are located in the loop regions of the duplex and
can be used to elongate the stem regions if desired.
[0468] The sequence of the test trigger oligonucleotide is shown in
FIG. 20B; this oligonucleotide is termed the alpha primer (SEQ ID
NO:25). The alpha primer is complementary to the 3' arm of the
T-hairpin as shown in FIG. 20A. When the alpha primer is annealed
to the T-hairpin, a cleavage structure is formed that is recognized
by thermostable DNA polymerases. Cleavage of the T-hairpin
liberates the 5' single-stranded arm of the T-hairpin, generating
the tau primer (SEQ ID NO:26) and a cleaved T-hairpin (FIG. 20B;
SEQ ID NO:27). The tau primer is complementary to the 3' arm of the
A-hairpin as shown in FIG. 20A. Annealing of the tau primer to the
A-hairpin generates another cleavage structure; cleavage of this
second cleavage structure liberates the 5' single-stranded arm of
the A-hairpin, generating another molecule of the alpha primer
which then is annealed to another molecule of the T-hairpin.
Thermocycling releases the primers so they can function in
additional cleavage reactions. Multiple cycles of annealing and
cleavage are carried out. The products of the cleavage reactions
are primers and the shortened hairpin structures shown in FIG. 20C.
The shortened or cleaved hairpin structures may be resolved from
the uncleaved hairpins by electrophoresis on denaturing acrylamide
gels.
[0469] The annealing and cleavage reactions are carried as follows:
In a 50 .mu.l reaction volume containing 10 mM Tris-Cl, pH 8.5, 1.0
MgCl.sub.2, 75 mM KCl, I pmole of A-hairpin, 1 pmole T-hairpin, the
alpha primer is added at equimolar amount relative to the hairpin
structures (1 pmole) or at dilutions ranging from 10- to
10.sup.6-fold and 0.5 .mu.l of extract containing a 5' nuclease
(prepared as described above) are added. The predicted melting
temperature for the alpha or trigger primer is 60.degree. C. in the
above buffer. Annealing is performed just below this predicted
melting temperature at 55.degree. C. Using a Perkin Elmer DNA
Thermal Cycler, the reactions are annealed at 55.degree. C. for 30
seconds. The temperature is then increased slowly over a five
minute period to 72.degree. C. to allow for cleavage. After
cleavage, the reactions are rapidly brought to 55.degree. C.
(1.degree. C. per second) to allow another cycle of annealing to
occur. A range of cycles are performed (20, 40 and 60 cycles) and
the reaction products are analyzed at each of these number of
cycles. The number of cycles which indicates that the accumulation
of cleaved hairpin products has not reached a plateau is then used
for subsequent determinations when it is desirable to obtain a
quantitative result.
[0470] Following the desired number of cycles, the reactions are
stopped at 55.degree. C. by the addition of 8 .mu.l of 95%
formamide with 20 mM EDTA and 0.05% marker dyes per 10 .mu.l
reaction volume. Samples are not heated before loading onto
denaturing polyacrylamide gels (10% polyacrylamide, 19:1
crosslinking, 7 M urea, 89 mM tris-borate, pH 8.3, 2.8 mM EDTA).
The samples were not heated to allow for the resolution of
single-stranded and re-duplexed uncleaved hairpin molecules.
[0471] The hairpin molecules may be attached to separate solid
support molecules, such as agarose, styrene or magnetic beads, via
the 3' end of each hairpin. A spacer molecule may be placed between
the 3' end of the hairpin and the bead if so desired. The advantage
of attaching the hairpins to a solid support is that this prevents
the hybridization of the A- and T-hairpins to one another during
the cycles of melting and annealing. The A- and T-hairpins are
complementary to one another (as shown in FIG. 20D) and if allowed
to anneal to one another over their entire lengths this would
reduce the amount of hairpins available for hybridization to the
alpha and tau primers during the detection reaction.
[0472] The 5' nucleases of the present invention are used in this
assay because they lack significant synthetic activity. The lack of
synthetic activity results in the production of a single cleaved
hairpin product (as shown in FIG. 19B, lane 4). Multiple cleavage
products may be generated by 1) the presence of interfering
synthetic activity (see FIG. 19B, lanes 6 and 8) or 2) the presence
of primer-independent cleavage in the reaction. The presence of
primer-independent cleavage is detected in the trigger/detection
assay by the presence of different sized products at the fork of
the cleavage structure. Primer-independent cleavage can be dampened
or repressed, when present, by the use of uncleavable nucleotides
in the fork region of the hairpin molecule. For example, thiolated
nucleotides can be used to replace several nucleotides at the fork
region to prevent primer-independent cleavage.
Example 5
Cleavage of Linear Nucleic Acid Substrates
[0473] From the above, it should be clear that native (i.e., "wild
type") thermostable DNA polymerases are capable of cleaving hairpin
structures in a specific manner and that this discovery can be
applied with success to a detection assay. In this example, the
mutant DNAPs of the present invention are tested against three
different cleavage structures shown in FIG. 22A. Structure 1 in
FIG. 22A is simply single stranded 206-mer (the preparation and
sequence information for which was discussed above). Structures 2
and 3 are duplexes; structure 2 is the same hairpin structure as
shown in FIG. 12A (bottom), while structure 3 has the hairpin
portion of structure 2 removed.
[0474] The cleavage reactions comprised 0.01 pmoles of the
resulting substrate DNA, and 1 pmole of pilot oligonucleotide in a
total volume of 10 .mu.l of 10 mM Tris-Cl, pH 8.3, 100 mM KCl, 1 mM
MgCl.sub.2. Reactions were incubated for 30 minutes at 55.degree.
C., and stopped by the addition of 8 t of 95% formamide with 20 mM
EDTA and 0.05% marker dyes. Samples were heated to 75.degree. C.
for 2 minutes immediately before electrophoresis through a 10%
polyacrylamide gel (19:1 cross link), with 7M urea, in a buffer of
45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.
[0475] The results were visualized by autoradiography and are shown
in FIG. 22B with the enzymes indicated as follows: I is native Taq
DNAP; II is native Tfl DNAP; III is Cleavase.RTM. BX shown in FIG.
4E; IV is Cleavase.RTM. BB shown in FIG. 4F; V is the mutant shown
in FIG. 5B; and VI is Cleavase.RTM. BN shown in FIG. 4G.
[0476] Structure 2 was used to "normalize" the comparison. For
example, it was found that it took 50 ng of Taq DNAP and 300 ng of
Cleavase.RTM. BN to give similar amounts of cleavage of Structure 2
in thirty (30) minutes. Under these conditions native Taq DNAP is
unable to cleave Structure 3 to any significant degree. Native Tfl
DNAP cleaves Structure 3 in a manner that creates multiple
products.
[0477] By contrast, all of the mutants tested cleave the linear
duplex of Structure 3. This finding indicates that this
characteristic of the mutant DNA polymerases is consistent of
thermostable polymerases across thermophilic species.
[0478] The finding described herein that the mutant DNA polymerases
of the present invention are capable of cleaving linear duplex
structures allows for application to a more straightforward assay
design (FIG. 1A). FIG. 23 provides a more detailed schematic
corresponding to the assay design of FIG. 1A.
[0479] The two 43-mers depicted in FIG. 23 were synthesized by
standard methods. Each included a fluorescein on the 5'end for
detection purposes and a biotin on the 3' end to allow attachment
to streptavidin coated paramagnetic particles (the biotin-avidin
attachment is indicated by " ").
[0480] Before the trityl groups were removed, the oligos were
purified by HPLC to remove truncated by-products of the synthesis
reaction. Aliquots of each 43-mer were bound to M-280 Dynabeads
(Dynal) at a density of 100 pmoles per mg of beads. Two (2) mgs of
beads (200 .mu.l) were washed twice in 1.times. wash/bind buffer (1
M NaCl, 5 mM Tris-Cl, pH 7.5, 0.5 mM EDTA) with 0.1% BSA, 200 .mu.l
per wash. The beads were magnetically sedimented between washes to
allow supernatant removal. After the second wash, the beads were
resuspended in 200 .mu.l of 2.times. wash/bind buffer (2 M Na Cl,
10 mM Tris-Cl, pH 7.5 with 1 mM EDTA), and divided into two 100
.mu.l aliquots. Each aliquot received 1 .mu.l of a 100 .mu.M
solution of one of the two oligonucleotides. After mixing, the
beads were incubated at room temperature for 60 minutes with
occasional gentle mixing. The beads were then sedimented and
analysis of the supernatants showed only trace amounts of unbound
oligonucleotide, indicating successful binding. Each aliquot of
beads was washed three times, 100 .mu.l per wash, with 1.times.
wash/bind buffer, then twice in a buffer of 10 mM Tris-Ci, pH 8.3
and 75 mM KCl. The beads were resuspended in a final volume of 100
.mu.l of the Tris/KCl, for a concentration of 1 pmole of oligo
bound to 10 .mu.g of beads per .mu.l of suspension. The beads were
stored at 4.degree. C. between uses.
[0481] The types of beads correspond to FIG. 1A. That is to say,
type 2 beads contain the oligo (SEQ ID NO:33) comprising the
complementary sequence (SEQ ID NO:34) for the alpha signal oligo
(SEQ ID NO:35) as well as the beta signal oligo (SEQ ID NO:36)
which when liberated is a 24-mer. This oligo has no "As" and is "T"
rich. Type 3 beads contain the oligo (SEQ ID NO:37) comprising the
complementary sequence (SEQ ID NO:38) for the beta signal oligo
(SEQ ID NO:39) as well as the alpha signal oligo (SEQ ID NO:35)
which when liberated is a 20-mer. This oligo has no "Ts" and is "A"
rich.
[0482] Cleavage reactions comprised 1 .mu.l of the indicated beads,
10 pmoles of unlabelled alpha signal oligo as "pilot" (if
indicated) and 500 ng of Cleavase.RTM. BN in 20 .mu.l of 75 mM KCl,
10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl.sub.2 and 10 .mu.M CTAB. All
components except the enzyme were assembled, overlaid with light
mineral oil and warmed to 53.degree. C. The reactions were
initiated by the addition of prewarmed enzyme and incubated at that
temperature for 30 minutes. Reactions were stopped at temperature
by the addition of 16 .mu.l of 95% formamide with 20 mM EDTA and
0.05% each of bromophenol blue and xylene cyanol. This addition
stops the enzyme activity and, upon heating, disrupts the
biotin-avidin link, releasing the majority (greater than 95%) of
the oligos from the beads. Samples were heated to 75.degree. C. for
2 minutes immediately before electrophoresis through a 10%
polyacrylamide gel (19:1 cross link), with 7 M urea, in a buffer of
45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Results were visualized by
contact transfer of the resolved DNA to positively charged nylon
membrane and probing of the blocked membrane with an
anti-fluorescein antibody conjugated to alkaline phosphatase. After
washing, the signal was developed by incubating the membrane in
Western Blue (Promega) which deposits a purple precipitate where
the antibody is bound.
[0483] FIG. 24 shows the propagation of cleavage of the linear
duplex nucleic acid structures of FIG. 23 by the DNAP mutants of
the present invention. The two center lanes contain both types of
beads. As noted above, the beta signal oligo (SEQ ID NO:36) when
liberated is a 24-mer and the alpha signal oligo (SEQ ID NO:35)
when liberated is a 20-mer. The formation of the two lower bands
corresponding to the 24-mer and 20-mer is clearly dependent on
"pilot".
Example 6
5' Exonucleolytic Cleavage ("Nibbling") by Thermostable DNAPs
[0484] It has been found that thermostable DNAPs, including those
of the present invention, have a true 5' exonuclease capable of
nibbling the 5' end of a linear duplex nucleic acid structures. In
this example, the 206 base pair DNA duplex substrate is again
employed (see above). In this case, it was produced by the use of
one .sup.32P-labeled primer and one unlabeled primer in a
polymerase chain reaction. The cleavage reactions comprised 0.01
pmoles of heat-denatured, end-labeled substrate DNA (with the
unlabeled strand also present), 5 pmoles of pilot oligonucleotide
(see pilot oligos in FIG. 12A) and 0.5 units of DNAPTaq or 0.5.mu.
of Cleavase.RTM. BB in the E. coli extract (see above), in a total
volume of 10 .mu.l of 10 mM Tris-Cl, pH 8.5, 50 mM KCl, 1.5 mM
MgCl.sub.2.
[0485] Reactions were initiated at 65.degree. C. by the addition of
pre-warmed enzyme, then shifted to the final incubation temperature
for 30 minutes. The results are shown in FIG. 25A. Samples in lanes
1-4 are the results with native Taq DNAP, while lanes 5-8 shown the
results with Cleavase.RTM. BB. The reactions for lanes 1, 2, 5, and
6 were performed at 65.degree. C. and reactions for lanes 3, 4, 7,
and 8 were performed at 50.degree. C. and all were stopped at
temperature by the addition of 8 .mu.l of 95% formamide with 20 mM
EDTA and 0.05% marker dyes. Samples were heated to 75.degree. C.
for 2 minutes immediately before electrophoresis through a 10%
acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of
45 mM Tris.Borate, pH 8.3, 1.4 mM EDTA. The expected product in
reactions 1, 2, 5, and 6 is 85 nucleotides long; in reactions 3 and
7, the expected product is 27 nucleotides long. Reactions 4 and 8
were performed without pilot, and should remain at 206 nucleotides.
The faint band seen at 24 nucleotides is residual end-labeled
primer from the PCR.
[0486] The surprising result is that Cleavase.RTM. BB under these
conditions causes all of the label to appear in a very small
species, suggesting the possibility that the enzyme completely
hydrolyzed the substrate. To determine the composition of the
fastest-migrating band seen in lanes 5-8 (reactions performed with
the deletion mutant), samples of the 206 base pair duplex were
treated with either T7 gene 6 exonuclease (USB) or with calf
intestine alkaline phosphatase (Promega), according to
manufacturers' instructions, to produce either labeled
mononucleotide (lane a of FIG. 25B) or free .sup.32P-labeled
inorganic phosphate (lane b of FIG. 25B), respectively. These
products, along with the products seen in lane 7 of panel A were
resolved by brief electrophoresis through a 20% acrylamide gel
(19:1 cross-link), with 7 M urea, in a buffer of 45 mM Tris.Borate,
pH 8.3, 1.4 mM EDTA. Cleavase.RTM. BB is thus capable of converting
the substrate to mononucleotides.
Example 7
Nibbling is Duplex Dependent
[0487] The nibbling by Cleavase.RTM. BB is duplex dependent. In
this example, internally labeled, single strands of the 206-mer
were produced by 15 cycles of primer extension incorporating
.alpha.-.sup.32P labeled dCTP combined with all four unlabeled
dNTPs, using an unlabeled 206-bp fragment as a template. Single and
double stranded products were resolved by electrophoresis through a
non-denaturing 6% polyacrylamide gel (29:1 cross-link) in a buffer
of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, visualized by
autoradiography, excised from the gel, eluted by passive diffusion,
and concentrated by ethanol precipitation.
[0488] The cleavage reactions comprised 0.04 pmoles of substrate
DNA, and 2 .mu.l of Cleavase.RTM. BB (in an E. coli extract as
described above) in a total volume of 40 .mu.l of 10 mM Tris.Cl, pH
8.5, 50 mM KCl, 1.5 mM MgCl.sub.2. Reactions were initiated by the
addition of pre-warmed enzyme; 10 .mu.l aliquots were removed at 5,
10, 20, and 30 minutes, and transferred to prepared tubes
containing 8 ill of 95% formamide with 30 mM EDTA and 0.05% marker
dyes. Samples were heated to 75.degree. C. for 2 minutes
immediately before electrophoresis through a 10% acrylamide gel
(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM
Tris-Borate, pH 8.3, 1.4 mM EDTA. Results were visualized by
autoradiography as shown in FIG. 26. Clearly, the cleavage by
Cleavase.RTM. BB depends on a duplex structure; no cleavage of the
single strand structure is detected whereas cleavage of the 206-mer
duplex is complete.
Example 8
Nibbling can be Target Directed
[0489] The nibbling activity of the DNAPs of the present invention
can be employed with success in a detection assay. One embodiment
of such an assay is shown in FIG. 27. In this assay, a labelled
oligo is employed that is specific for a target sequence. The oligo
is in excess of the target so that hybridization is rapid. In this
embodiment, the oligo contains two fluorescein labels whose
proximity on the oligo causes their emission to be quenched. When
the DNAP is permitted to nibble the oligo the labels separate and
are detectable. The shortened duplex is destabilized and
disassociates. Importantly, the target is now free to react with an
intact labelled oligo. The reaction can continue until the desired
level of detection is achieved. An analogous, although different,
type of cycling assay has been described employing lambda
exonuclease. See C. G. Copley and C. Boot, BioTechniques 13:888
(1992).
[0490] The success of such an assay depends on specificity. In
other words, the oligo must hybridize to the specific target. It is
also preferred that the assay be sensitive; the oligo ideally
should be able to detect small amounts of target. FIG. 28A shows a
5'-end .sup.32P-labelled primer bound to a plasmid target sequence.
In this case, the plasmid was pUC19 (commercially available) which
was heat denatured by boiling two (2) minutes and then quick
chilling. The primer is a 21-mer (SEQ ID NO:39). The enzyme
employed was Cleavase.RTM. BX (a dilution equivalent to
5.times.10.sup.-3 .mu.l extract) in 100 mM KCl, 10 mM Tris-Cl, pH
8.3, 2 mM MnCl.sub.2. The reaction was performed at 55.degree. C.
for sixteen (16) hours with or without genomic background DNA (from
chicken blood). The reaction was stopped by the addition of 8 .mu.l
of 95% formamide with 20 mM EDTA and marker dyes.
[0491] The products of the reaction were resolved by PAGE (10%
polyacrylamide, 19:1 cross link, 1.times.TBE) as seen in FIG. 28B.
Lane "M" contains the labelled 21-mer. Lanes 1-3 contain no
specific target, although Lanes 2 and 3 contain 100 ng and 200 ng
of genomic DNA, respectively. Lanes 4, 5 and 6 all contain specific
target with either 0 ng, 100 ng or 200 ng of genomic DNA,
respectively. It is clear that conversion to mononucleotides occurs
in Lanes 4, 5 and 6 regardless of the presence or amount of
background DNA. Thus, the nibbling can be target directed and
specific.
Example 9
Cleavase Purification
[0492] As noted above, expressed thermostable proteins, i.e., the
5' nucleases, were isolated by crude bacterial cell extracts. The
precipitated E. coli proteins were then, along with other cell
debris, removed by centrifugation. In this example, cells
expressing the BN clone were cultured and collected (500 grams).
For each gram (wet weight) of E. coli, 3 ml of lysis buffer (50 mM
Tris-HCl, pH 8.0, 1 mM EDTA, 100 .mu.M NaCl) was added. The cells
were lysed with 200 .mu.g/ml lysozyme at room temperature for 20
minutes. Thereafter deoxycholic acid was added to make a 0.2% final
concentration and the mixture was incubated 15 minutes at room
temperature.
[0493] The lysate was sonicated for approximately 6-8 minutes at
0.degree. C. The precipitate was removed by centrifugation (39,000
g for 20 minutes). Polyethyleneimine was added (0.5%) to the
supernatant and the mixture was incubated on ice for 15 minutes.
The mixture was centrifuged (5,000 g for 15 minutes) and the
supernatant was retained. This was heated for 30 minutes at
60.degree. C. and then centrifuged again (5,000 g for 15 minutes)
and the supernatant was again retained.
[0494] The supernatant was precipitated with 35% ammonium sulfate
at 4.degree. C. for 15 minutes. The mixture was then centrifuged
(5,000 g for 15 minutes) and the supernatant was removed. The
precipitate was then dissolved in 0.25 M KCl, 20 Tris pH 7.6, 0.2%
Tween and 0.1 EDTA) and then dialyzed against Binding Buffer
(8.times. Binding Buffer comprises: 40 mM imidazole, 4M NaCl, 160
mM Tris-HCl, pH 7.9).
[0495] The solubilized protein is then purified on the Ni.sup.++
column (Novagen). The Binding Buffer is allows to drain to the top
of the column bed and load the column with the prepared extract. A
flow rate of about 10 column volumes per hour is optimal for
efficient purification. If the flow rate is too fast, more
impurities will contaminate the eluted fraction.
[0496] The column is washed with 25 ml (10 volumes) of 1.times.
Binding Buffer and then washed with 15 ml (6 volumes) of 1.times.
Wash Buffer (8.times. Wash Buffer comprises: 480 mM imidazole, 4M
NaCl, 160 mM Tris-HCl, pH 7.9). The bound protein was eluted with
15 ml (6 volumes) of 1.times. Elute Buffer (4.times. Elute Buffer
comprises: 4 mM imidazole, 2 M NaCl, 80 mM Tris-HCl, pH 7.9).
Protein is then reprecipitated with 35% Ammonium Sulfate as above.
The precipitate was then dissolved and dialyzed against: 20 mM
Tris, 100 mM KCl, 1 mM EDTA). The solution was brought up to 0.1%
each of Tween 20 and NP-40 and stored at 4.degree. C.
Example 10
The Use of Various Divalent Cations in the Cleavage Reaction
Influences the Nature of the Resulting Cleavage Products
[0497] In comparing the 5' nucleases generated by the modification
and/or deletion of the C-terminal polymerization domain of Thermus
aquaticus DNA polymerase (DNAPTaq), as diagrammed in FIG. 4B-G,
significant differences in the strength of the interactions of
these proteins with the 3' end of primers located upstream of the
cleavage site (as depicted in FIG. 6) were noted. In describing the
cleavage of these structures by Pol l-type DNA polymerases [Example
1 and Lyamichev et al. (1993) Science 260:778], it was observed
that in the absence of a primer, the location of the junction
between the double-stranded region and the single-stranded 5' and
3' arms determined the site of cleavage, but in the presence of a
primer, the location of the 3' end of the primer became the
determining factor for the site of cleavage. It was postulated that
this affinity for the 3' end was in accord with the synthesizing
function of the DNA polymerase.
[0498] Structure 2, shown in FIG. 22A, was used to test the effects
of a 3' end proximal to the cleavage site in cleavage reactions
comprising several different solutions [e.g., solutions containing
different salts (KCl or NaCl), different divalent cations
(Mn.sup.2+ or Me.sup.2+), etc.] as well as the use of different
temperatures for the cleavage reaction. When the reaction
conditions were such that the binding of the enzyme (e.g., a DNAP
comprising a 5' nuclease, a modified DNAP or a 5' nuclease) to the
3' end (of the pilot oligonucleotide) near the cleavage site was
strong, the structure shown is cleaved at the site indicated in
FIG. 22A. This cleavage releases the unpaired 5' arm and leaves a
nick between the remaining portion of the target nucleic acid and
the folded 3' end of the pilot oligonucleotide. In contrast, when
the reaction conditions are such that the binding of the DNAP
(comprising a 5' nuclease) to the 3' end was weak, the initial
cleavage was as described above, but after the release of the 5'
arm, the remaining duplex is digested by the exonuclease function
of the DNAP.
[0499] One way of weakening the binding of the DNAP to the 3' end
is to remove all or part of the domain to which at least some of
this function has been attributed. Some of 5' nucleases created by
deletion of the polymerization domain of DNAPTaq have enhanced true
exonuclease function, as demonstrated in Example 6.
[0500] The affinity of these types of enzymes (i.e., 5' nucleases
associated with or derived from DNAPs) for recessed 3' ends may
also be affected by the identity of the divalent cation present in
the cleavage reaction. It was demonstrated by Longley et al. [Nucl.
Acids Res. 18:7317 (1990)] that the use of MnCl.sub.2 in a reaction
with DNAPTaq enabled the polymerase to remove nucleotides from the
5' end of a primer annealed to a template, albeit inefficiently.
Similarly, by examination of the cleavage products generated using
Structure 2 from FIG. 22A, as described above, in a reaction
containing either DNAPTaq or the Cleavase.RTM. BB nuclease, it was
observed that the substitution of MnCl.sub.2 for MgCl.sub.2 in the
cleavage reaction resulted in the exonucleolytic "nibbling" of the
duplex downstream of the initial cleavage site. While not limiting
the invention to any particular mechanism, it is thought that the
substitution of MnCl.sub.2 for MgCl.sub.2 in the cleavage reaction
lessens the affinity of these enzymes for recessed 3' ends.
[0501] In all cases, the use of MnCl.sub.2 enhances the 5' nuclease
function, and in the case of the Cleavase.RTM. BB nuclease, a 50-
to 100-fold stimulation of the 5' nuclease function is seen. Thus,
while the exonuclease activity of these enzymes was demonstrated
above in the presence of MgCl.sub.2, the assays described below
show a comparable amount of exonuclease activity using 50 to
100-fold less enzyme when MnCl.sub.2 is used in place of
MgCl.sub.2. When these reduced amounts of enzyme are used in a
reaction mixture containing MgCl.sub.2, the nibbling or exonuclease
activity is much less apparent than that seen in Examples 6-8.
[0502] Similar effects are observed in the performance of the
nucleic acid detection assay described in Examples 11-18 below when
reactions performed in the presence of either MgCl.sub.2 or
MnCl.sub.2 are compared. In the presence of either divalent cation,
the presence of the invader oligonucleotide (described below)
forces the site of cleavage into the probe duplex, but in the
presence of MnCl.sub.2 the probe duplex can be further nibbled
producing a ladder of products that are visible when a 3' end label
is present on the probe oligonucleotide. When the invader
oligonucleotide is omitted from a reaction containing Mn.sup.2+,
the probe is nibbled from the 5' end. Mg.sup.2+-based reactions
display minimal nibbling of the probe oligonucleotide. In any of
these cases, the digestion of the probe is dependent upon the
presence of the target nucleic acid. In the examples below, the
ladder produced by the enhanced nibbling activity observed in the
presence of Mn.sup.2+ is used as a positive indicator that the
probe oligonucleotide has hybridized to the target sequence.
Example 11
Invasive 5' Endonucleolytic Cleavage by Thermostable 5' Nucleases
in the Absence of Polymerization
[0503] As described in the examples above, 5' nucleases cleave near
the junction between single-stranded and base-paired regions in a
bifurcated duplex, usually about one base pair into the base-paired
region. In this example, it is shown that thermostable 5'
nucleases, including those of the present invention (e.g.
Cleavase.RTM. BN nuclease, Cleavase.RTM. A/G nuclease), have the
ability to cleave a greater distance into the base paired region
when provided with an upstream oligonucleotide bearing a 3' region
that is homologous to a 5' region of the subject duplex, as shown
in FIG. 30.
[0504] FIG. 30 shows a synthetic oligonucleotide which was designed
to fold upon itself which consists of the following sequence:
5'-GTTCTCTGCTCTCTGGTCGCTG TCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3'
(SEQ ID NO:40). This oligonucleotide is referred to as the "S-60
Hairpin." The 15 basepair hairpin formed by this oligonucleotide is
further stabilized by a "tri-loop" sequence in the loop end (i.e.,
three nucleotides form the loop portion of the hairpin) [Hiraro, I.
et al. (1994) Nucleic Acids Res. 22(4):576]. FIG. 30 also show the
sequence of the P-15 oligonucleotide and the location of the region
of complementarity shared by the P-15 and S-60 hairpin
oligonucleotides. The sequence of the P-15 oligonucleotide is
5'-CGAGAGACCACGCTG-3' (SEQ ID NO:41). As discussed in detail below,
the solid black arrowheads shown in FIG. 29 indicate the sites of
cleavage of the S-60 hairpin in the absence of the P-15
oligonucleotide and the hollow arrow heads indicate the sites of
cleavage in the presence of the P-15 oligonucleotide. The size of
the arrow head indicates the relative utilization of a particular
site.
[0505] The S-60 hairpin molecule was labeled on its 5' end with
biotin for subsequent detection. The S-60 hairpin was incubated in
the presence of a thermostable 5' nuclease in the presence or the
absence of the P-15 oligonucleotide. The presence of the full
duplex which can be formed by the S-60 hairpin is demonstrated by
cleavage with the Cleavase.RTM. BN 5' nuclease, in a
primer-independent fashion (i.e., in the absence of the P-15
oligonucleotide). The release of 18 and 19-nucleotide fragments
from the 5' end of the S-60 hairpin molecule showed that the
cleavage occurred near the junction between the single and double
stranded regions when nothing is hybridized to the 3' arm of the
S-60 hairpin (FIG. 31, lane 2).
[0506] The reactions shown in FIG. 31 were conducted as follows.
Twenty fmole of the 5' biotin-labeled hairpin DNA (SEQ ID NO:40)
was combined with 0.1 ng of Cleavase.RTM. BN enzyme and 141 of 100
mM MOPS (pH 7.5) containing 0.5% each of Tween-20 and NP-40 in a
total volume of 9 .mu.l. In the reaction shown in lane 1, the
enzyme was omitted and the volume was made up by addition of
distilled water (this served as the uncut or no enzyme control).
The reaction shown in lane 3 of FIG. 31 also included 0.5 pmole of
the P15 oligonucleotide (SEQ ID NO:41), which can hybridize to the
unpaired 3' arm of the S-60 hairpin (SEQ ID NO:40), as diagrammed
in FIG. 30.
[0507] The reactions were overlaid with a drop of mineral oil,
heated to 95.degree. C. for 15 seconds, then cooled to 37.degree.
C., and the reaction was started by the addition of 1 .mu.l of 10
mM MnCl.sub.2 to each tube. After 5 minutes, the reactions were
stopped by the addition of 6 .mu.l of 95% formamide containing 20
mM EDTA and 0.05% marker dyes. Samples were heated to 75.degree. C.
for 2 minutes immediately before electrophoresis through a 15%
acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of
45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.
[0508] After electrophoresis, the gel plates were separated
allowing the gel to remain flat on one plate. A 0.2 mm-pore
positively-charged nylon membrane (NYTRAN, Schleicher and Schuell,
Keene, N.H.), pre-wetted in H.sub.2O, was laid on top of the
exposed gel. All air bubbles were removed. Two pieces of 3MM filter
paper (Whatman) were then placed on top of the membrane, the other
glass plate was replaced, and the sandwich was clamped with binder
clips. Transfer was allowed to proceed overnight. After transfer,
the membrane was carefully peeled from the gel and allowed to air
dry. After complete drying, the membrane was washed in 1.2.times.
Sequenase Images Blocking Buffer (United States Biochemical) using
0.3 ml of buffer/cm.sup.2 of membrane. The wash was performed for
30 minutes at room temperature. A streptavidin-alkaline phosphatase
conjugate (SAAP, United States Biochemical) was added to a 1:4000
dilution directly to the blocking solution, and agitated for 15
minutes. The membrane was rinsed briefly with H.sub.2O and then
washed three times for 5 minutes per wash using 0.5 ml/cm.sup.2 of
1.times. SAAP buffer (100 mM Tris-HCl, pH 10, 50 mM NaCl) with 0.1%
sodium dodecyl sulfate (SDS). The membrane was rinsed briefly with
H.sub.2O between each wash. The membrane was then washed once in
1.times. SAAP buffer containing 1 mM MgCl.sub.2 without SDS,
drained thoroughly and placed in a plastic heat-sealable bag. Using
a sterile pipet, 5 mls of CDP-Star.TM. (Tropix, Bedford, Mass.)
chemiluminescent substrate for alkaline phosphatase were added to
the bag and distributed over the entire membrane for 2-3 minutes.
The CDP-Star.TM.-treated membrane was exposed to XRP X-ray film
(Kodak) for an initial exposure of 10 minutes.
[0509] The resulting autoradiograph is shown in FIG. 31. In FIG.
31, the lane labelled "M" contains the biotinylated P-15
oligonucleotide which served as a marker. The sizes (in
nucleotides) of the uncleaved S-60 hairpin (60 nuc; lane 1), the
marker (15 nuc; lane "M") and the cleavage products generated by
cleavage of the S-60 hairpin in the presence (lane 3) or absence
(lane 2) of the P-15 oligonucleotide are indicated.
[0510] Because the complementary regions of the S-60 hairpin are
located on the same molecule, essentially no lag time should be
needed to allow hybridization (i.e., to form the duplex region of
the hairpin). This hairpin structure would be expected to form long
before the enzyme could locate and cleave the molecule. As
expected, cleavage in the absence of the primer oligonucleotide was
at or near the junction between the duplex and single-stranded
regions, releasing the unpaired 5' arm (FIG. 31, lane 2). The
resulting cleavage products were 18 and 19 nucleotides in
length.
[0511] It was expected that stability of the S-60 hairpin with the
tri-loop would prevent the P-15 oligonucleotide from promoting
cleavage in the "primer-directed" manner described in Example 1
above, because the 3' end of the "primer" would remain unpaired.
Surprisingly, it was found that the enzyme seemed to mediate an
"invasion" by the P-15 primer into the duplex region of the S-60
hairpin, as evidenced by the shifting of the cleavage site 3 to 4
basepairs further into the duplex region, releasing the larger
products (22 and 21 nuc.) observed in lane 3 of FIG. 31.
[0512] The precise sites of cleavage of the S-60 hairpin are
diagrammed on the structure in FIG. 30, with the solid black
arrowheads indicating the sites of cleavage in the absence of the
P-15 oligonucleotide and the hollow arrow heads indicating the
sites of cleavage in the presence of P-15.
[0513] These data show that the presence on the 3' arm of an
oligonucleotide having some sequence homology with the first
several bases of the similarly oriented strand of the downstream
duplex can be a dominant factor in determining the site of cleavage
by 5' nucleases. Because the oligonucleotide which shares some
sequence homology with the first several bases of the similarly
oriented strand of the downstream duplex appears to invade the
duplex region of the hairpin, it is referred to as an" invader"
oligonucleotide. As shown in the examples below, an invader
oligonucleotide appears to invade (or displace) a region of
duplexed nucleic acid regardless of whether the duplex region is
present on the same molecule (i.e., a hairpin) or whether the
duplex is formed between two separate nucleic acid strands.
Example 12
The Invader Oligonucleotide Shifts the Site Of Cleavage in a
Pre-Formed Probe/Target Duplex
[0514] In Example 11 it was demonstrated that an invader
oligonucleotide could shift the site at which a 5' nuclease cleaves
a duplex region present on a hairpin molecule. In this example, the
ability of an invader oligonucleotide to shift the site of cleavage
within a duplex region formed between two separate strands of
nucleic acid molecules was examined.
[0515] A single-stranded target DNA comprising the single-stranded
circular M13mp19 molecule and a labeled (fluorescein) probe
oligonucleotide were mixed in the presence of the reaction buffer
containing salt (KCl) and divalent cations (Mg.sup.2+ or M.sup.2+)
to promote duplex formation. The probe oligonucleotide refers to a
labelled oligonucleotide which is complementary to a region along
the target molecule (e.g., M13 mp19). A second oligonucleotide
(unlabelled) was added to the reaction after the probe and target
had been allowed to anneal. The second oligonucleotide binds to a
region of the target which is located downstream of the region to
which the probe oligonucleotide binds. This second oligonucleotide
contains sequences which are complementary to a second region of
the target molecule. If the second oligonucleotide contains a
region which is complementary to a portion of the sequences along
the target to which the probe oligonucleotide also binds, this
second oligonucleotide is referred to as an invader oligonucleotide
(see FIG. 32c).
[0516] FIG. 32 depicts the annealing of two oligonucleotides to
regions along the M13 mp19 target molecule (bottom strand in all
three structures shown). In FIG. 32 only a 52 nucleotide portion of
the M13 mp19 molecule is shown; this 52 nucleotide sequence is
listed in SEQ ID NO:42. The probe oligonucleotide contains a
fluorescein label at the 3' end; the sequence of the probe is
5'-AGAAAGGAAGGGAAGAAAGC GAAAGG-3' (SEQ ID NO:43). In FIG. 32,
sequences comprising the second oligonucleotide, including the
invader oligonucleotide are underlined. In FIG. 32a, the second
oligonucleotide, which has the sequence 5'-GACGGGGAAAGCCGGCGA
ACG-3' (SEQ ID NO:44), is complementary to a different and
downstream region of the target molecule than is the probe
oligonucleotide (labeled with fluorescein or "Fluor"); there is a
gap between the second, upstream oligonucleotide and the probe for
the structure shown in FIG. 32a. In FIG. 32b, the second, upstream
oligonucleotide, which has the sequence 5'-GAAAGCCGGCGAACGTGGCG-3'
(SEQ ID NO:45), is complementary to a different region of the
target molecule than is the probe oligonucleotide, but in this
case, the second oligonucleotide and the probe oligonucleotide abut
one another (that is the 3' end of the second, upstream
oligonucleotide is immediately adjacent to the 5' end of the probe
such that no gap exists between these two oligonucleotides). In
FIG. 32c, the second, upstream oligonucleotide
[5'-GGCGAACGTGGCGAGAAAGGA-3' (SEQ ID NO:46)] and the probe
oligonucleotide share a region of complementarity with the target
molecule. Thus, the upstream oligonucleotide has a 3' arm which has
a sequence identical to the first several bases of the downstream
probe. In this situation, the upstream oligonucleotide is referred
to as an "invader" oligonucleotide.
[0517] The effect of the presence of an invader oligonucleotide
upon the pattern of cleavage in a probe/target duplex formed prior
to the addition of the invader was examined. The invader
oligonucleotide and the enzyme were added after the probe was
allowed to anneal to the target and the position and extent of
cleavage of the probe were examined to determine a) if the invader
was able to shift the cleavage site to a specific internal region
of the probe, and b), if the reaction could accumulate specific
cleavage products over time, even in the absence of thermal
cycling, polymerization, or exonuclease removal of the probe
sequence.
[0518] The reactions were carried out as follows. Twenty .mu.l each
of two enzyme mixtures were prepared, containing 2 .mu.l of
Cleavase.RTM. A/G nuclease extract (prepared as described in
Example 2), with or without 50 pmole of the invader oligonucleotide
(SEQ ID NO:46), as indicated, per 4 .mu.l of the mixture. For each
of the eight reactions shown in FIG. 33, 150 fmole of M13 mp19
single-stranded DNA (available from Life Technologies, Inc.) was
combined with 5 pmoles of fluorescein labeled probe (SEQ ID NO:43),
to create the structure shown in FIG. 31c, but without the invader
oligonucleotide present (the probe/target mixture). One half (4
tubes) of the probe/target mixtures were combined with 1 .mu.l of
100 mM MOPS, pH 7.5 with 0.5% each of Tween-20 and NP-40, 0.5 .mu.l
of 1 M KCl and 0.25 .mu.l of 80 mM MnCl.sub.2, and distilled water
to a volume of 6 .mu.l. The second set of probe/target mixtures
were combined with 1 .mu.l of 100 mM MOPS, pH 7.5 with 0.5% each of
Tween-20 and NP-40, 0.5 .mu.l of 1 M KCl and 0.25 .mu.l of 80 mM
MgCl.sub.2. The second set of mixtures therefore contained
MgCl.sub.2 in place of the MnCl.sub.2 present in the first set of
mixtures.
[0519] The mixtures (containing the probe/target with buffer, KCl
and divalent cation) were covered with a drop of ChillOut.RTM.
evaporation barrier (MJ Research) and were brought to 60.degree. C.
for 5 minutes to allow annealing. Four .mu.l of the above enzyme
mixtures without the invader oligonucleotide was added to reactions
whose products are shown in lanes 1, 3, 5 and 7 of FIG. 33.
Reactions whose products are shown lanes 2, 4, 6, and 8 of FIG. 33
received the same amount of enzyme mixed with the invader
oligonucleotide (SEQ ID NO:46). Reactions 1, 2, 5 and 6 were
incubated for 5 minutes at 60.degree. C. and reactions 3, 4, 7 and
8 were incubated for 15 minutes at 60.degree. C.
[0520] All reactions were stopped by the addition of 8 .mu.l of 95%
formamide with 20 mM EDTA and 0.05% marker dyes. Samples were
heated to 90.degree. C. for 1 minute immediately before
electrophoresis through a 20% acrylamide gel (19:1 cross-linked),
containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4
mM EDTA. Following electrophoresis, the reaction products and were
visualized by the use of an Hitachi FMBIO fluorescence imager, the
output of which is seen in FIG. 33. The very low molecular weight
fluorescent material seen in all lanes at or near the salt front in
FIG. 33 and other fluoro-imager figures is observed when
fluorescently-labeled oligonucleotides are electrophoresed and
imaged on a fluoro-imager. This material is not a product of the
cleavage reaction.
[0521] The use of MnCl.sub.2 in these reactions (lanes 1-4)
stimulates the true exonuclease or "nibbling" activity of the
Cleavase.RTM. enzyme, as described in Example 7, as is clearly seen
in lanes 1 and 3 of FIG. 33. This nibbling of the probe
oligonucleotide (SEQ ID NO:43) in the absence of invader
oligonucleotide (SEQ ID NO:46) confirms that the probe
oligonucleotide is forming a duplex with the target sequence. The
ladder-like products produced by this nibbling reaction may be
difficult to differentiate from degradation of the probe by
nucleases that might be present in a clinical specimen. In
contrast, introduction of the invader oligonucleotide (SEQ ID
NO:46) caused a distinctive shift in the cleavage of the probe,
pushing the site of cleavage 6 to 7 bases into the probe,
confirming the annealing of both oligonucleotides. In presence of
MnCl.sub.2, the exonuclease "nibbling" may occur after the
invader-directed cleavage event, until the residual duplex is
destabilized and falls apart.
[0522] In a magnesium based cleavage reaction (lanes 5-8), the
nibbling or true exonuclease function of the Cleavase.RTM. A/G is
enzyme suppressed (but the endonucleolytic function of the enzyme
is essentially unaltered), so the probe oligonucleotide is not
degraded in the absence of the invader (FIG. 33, lanes 5 and 7).
When the invader is added, it is clear that the invader
oligonucleotide can promote a shift in the site of the
endonucleolytic cleavage of the annealed probe. Comparison of the
products of the 5 and 15 minute reactions with invader (lanes 6 and
8 in FIG. 33) shows that additional probe hybridizes to the target
and is cleaved. The calculated melting temperature (T.sub.m) of the
portion of probe that is not invaded (i.e., nucleotides 9-26 of SEQ
ID NO:43) is 56.degree. C., so the observed turnover (as evidenced
by the accumulation of cleavage products with increasing reaction
time) suggests that the full length of the probe molecule, with a
calculated T.sub.m of 76.degree. C., is must be involved in the
subsequent probe annealing events in this 60.degree. C.
reaction.
Example 13
[0523] The Overlap of the 3' Invader Oligonucleotide Sequence with
The 5' Region of the Probe Causes a Shift in the Site of
Cleavage
[0524] In Example 12, the ability of an invader oligonucleotide to
cause a shift in the site of cleavage of a probe annealed to a
target molecule was demonstrated. In this example, experiments were
conducted to examine whether the presence of an oligonucleotide
upstream from the probe was sufficient to cause a shift in the
cleavage site(s) along the probe or whether the presence of
nucleotides on the 3' end of the invader oligonucleotide which have
the same sequence as the first several nucleotides at the 5' end of
the probe oligonucleotide were required to promote the shift in
cleavage.
[0525] To examine this point, the products of cleavage obtained
from three different arrangements of target-specific
oligonucleotides are compared. A diagram of these oligonucleotides
and the way in which they hybridize to a test nucleic acid, M13
mp19, is shown in FIG. 32. In FIG. 32a, the 3' end of the upstream
oligonucleotide (SEQ ID NO:45) is located upstream of the 5' end of
the downstream "probe" oligonucleotide (SEQ ID NO:43) such that a
region of the M13 target which is not paired to either
oligonucleotide is present. In FIG. 32b, the sequence of the
upstream oligonucleotide (SEQ ID NO:45) is immediately upstream of
the probe (SEQ ID NO:43), having neither a gap nor an overlap
between the sequences. FIG. 32c diagrams the arrangement of the
substrates used in the assay of the present invention, showing that
the upstream "invader" oligonucleotide (SEQ ID NO:46) has the same
sequence on a portion of its 3' region as that present in the 5'
region of the downstream probe (SEQ ID NO:43). That is to say,
these regions will compete to hybridize to the same segment of the
M13 target nucleic acid.
[0526] In these experiments, four enzyme mixtures were prepared as
follows (planning 5 .mu.l per digest): Mixture 1 contained 2.25%1
of Cleavase.RTM. A/G nuclease extract (prepared as described in
Example 2) per 5 .mu.l of mixture, in 20 mM MOPS, pH 7.5 with 0.1%
each of Tween 20 and NP-40, 4 mM MnCl.sub.2 and 100 mM KCl. Mixture
2 contained 11.25 units of Taq DNA polymerase (Promega Corp.,
Madison, Wis.) per 5 .mu.l of mixture in 20 mM MOPS, pH 7.5 with
0.1% each of Tween 20 and NP-40, 4 mM MnCl.sub.2 and 100 mM KCl.
Mixture 3 contained 2.25 .mu.l of Cleavase.RTM. A/G nuclease
extract per 5 .mu.l of mixture in 20 mM Tris-HCl, pH 8.5, 4 mM
MgCl.sub.2 and 100 mM KCl. Mixture 4 contained 11.25 units of Taq
DNA polymerase per 5 .mu.l of mixture in 20 mM Tris-HCl, pH 8.5, 4
mM MgCl.sub.2 and 100 mM KCl.
[0527] For each reaction, 50 fmole of M13 mp19 single-stranded DNA
(the target nucleic acid) was combined with 5 pmole of the probe
oligonucleotide (SEQ ID NO:43 which contained a fluorescein label
at the 3' end) and 50 pmole of one of the three upstream
oligonucleotides diagrammed in FIG. 32 (i.e., one of SEQ ID
NOS:44-46), in a total volume of 5 .mu.l of distilled water. The
reactions were overlaid with a drop of ChillOut.TM. evaporation
barrier (MJ Research) and warmed to 62.degree. C. The cleavage
reactions were started by the addition of 5 .mu.l of an enzyme
mixture to each tube, and the reactions were incubated at
62.degree. C. for 30 min. The reactions shown in lanes 1-3 of FIG.
34 received Mixture 1; reactions 4-6 received Mixture 2; reactions
7-9 received Mixture 3 and reactions 10-12 received Mixture 4.
[0528] After 30 minutes at 62.degree. C., the reactions were
stopped by the addition of 8 .mu.l of 95% formamide with 20 mM EDTA
and 0.05% marker dyes. Samples were heated to 75.degree. C. for 2
minutes immediately before electrophoresis through a 20% acrylamide
gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM
Tris-Borate, pH 8.3, 1.4 mM EDTA.
[0529] Following electrophoresis, the products of the reactions
were visualized by the use of an Hitachi FMBIO fluorescence imager,
the output of which is seen in FIG. 34. The reaction products shown
in lanes 1, 4, 7 and 10 of FIG. 34 were from reactions which
contained SEQ ID NO:44 as the upstream oligonucleotide (see FIG.
32a). The reaction products shown in lanes 2, 5, 8 and 11 of FIG.
34 were from reactions which contained SEQ ID NO:45 as the upstream
oligonucleotide (see FIG. 32b). The reaction products shown in
lanes 3, 6, 9 and 12 of FIG. 34 were from reactions which contained
SEQ ID NO:46, the invader oligonucleotide, as the upstream
oligonucleotide (see FIG. 32c).
[0530] Examination of the Mn.sup.2+ based reactions using either
Cleavase.RTM. A/G nuclease or DNAPTaq as the cleavage agent (lanes
1 through 3 and 4 through 6, respectively) shows that both enzymes
have active exonuclease function in these buffer conditions. The
use of a 3' label on the probe oligonucleotide allows the products
of the nibbling activity to remain labeled, and therefore visible
in this assay. The ladders seen in lanes 1, 2, 4 and 5 confirm that
the probe hybridize to the target DNA as intended. These lanes also
show that the location of the non-invasive oligonucleotides have
little effect on the products generated. The uniform ladder created
by these digests would be difficult to distinguish from a ladder
causes by a contaminating nuclease, as one might find in a clinical
specimen. In contrast, the products displayed in lanes 3 and 6,
where an invader oligonucleotide was provided to direct the
cleavage, show a very distinctive shift, so that the primary
cleavage product is smaller than those seen in the non-invasive
cleavage. This product is then subject to further nibbling in these
conditions, as indicated by the shorter products in these lanes.
These invader-directed cleavage products would be easily
distinguished from a background of non-specific degradation of the
probe oligonucleotide.
[0531] When Mg.sup.+ is used as the divalent cation the results are
even more distinctive. In lanes 7, 8, 10 and 11 of FIG. 34, where
the upstream oligonucleotides were not invasive, minimal nibbling
is observed. The products in the DNAPTaq reactions show some
accumulation of probe that has been shortened on the 5' end by one
or two nucleotides consistent with previous examination of the
action of this enzyme on nicked substrates (Longley et al., supra).
When the upstream oligonucleotide is invasive, however, the
appearance of the distinctively shifted probe band is seen. These
data clearly indicated that it is the invasive 3' portion of the
upstream oligonucleotide that is responsible for fixing the site of
cleavage of the downstream probe.
[0532] Thus, the above results demonstrate that it is the presence
of the free or initially non-annealed nucleotides at the 3' end of
the invader oligonucleotide which mediate the shift in the cleavage
site, not just the presence of an oligonucleotide annealed upstream
of the probe. Nucleic acid detection assays which employ the use of
an invader oligonucleotide are termed "invader-directed cleavage"
assays.
Example 14
Invader-Directed Cleavage Recognizes Single and Double Stranded
Target Molecules in a Background of Non-Target DNA Molecules
[0533] For a nucleic acid detection method to be broadly useful, it
must be able to detect a specific target in a sample that may
contain large amounts of other DNA, e.g., bacterial or human
chromosomal DNA. The ability of the invader directed cleavage assay
to recognize and cleave either single- or double-stranded target
molecules in the presence of large amounts of non-target DNA was
examined. In these experiments a model target nucleic acid, M13, in
either single or double stranded form (single-stranded M13 mp18 is
available from Life Technologies, Inc and double-stranded M13 mp19
is available from New England Biolabs), was combined with human
genomic DNA (Novagen, Madison, Wis.) and then utilized in
invader-directed cleavage reactions. Before the start of the
cleavage reaction, the DNAs were heated to 95.degree. C. for 15
minutes to completely denature the samples, as is standard practice
in assays, such as polymerase chain reaction or enzymatic DNA
sequencing, which involve solution hybridization of
oligonucleotides to double-stranded target molecules.
[0534] For each of the reactions shown in lanes 2-5 of FIG. 35, the
target DNA (25 fmole of the ss DNA or 1 pmole of the ds DNA) was
combined with 50 pmole of the invader oligonucleotide (SEQ ID
NO:46); for the reaction shown in lane 1 the target DNA was
omitted. Reactions 1, 3 and 5 also contained 470 ng of human
genomic DNA. These mixtures were brought to a volume of 10 .mu.L
with distilled water, overlaid with a drop of ChillOut.TM.
evaporation barrier (MJ Research), and brought to 95.degree. C. for
15 minutes. After this incubation period, and still at 95.degree.
C., each tube received 10 .mu.l of a mixture comprising 2.25 .mu.l
of Cleavase.RTM. A/G nuclease extract (prepared as described in
Example 2) and 5 pmole of the probe oligonucleotide (SEQ ID NO:43),
in 20 mM MOPS, pH 7.5 with 0.1% each of Tween 20 and NP-40, 4 mM
MnCl.sub.2 and 100 mM KCl. The reactions were brought to 62.degree.
C. for 15 minutes and stopped by the addition of 12 .mu.l of 95%
formamide with 20 mM EDTA and 0.05% marker dyes. Samples were
heated to 75.degree. C. for 2 minutes immediately before
electrophoresis through a 20% acrylamide gel (19:1 cross-linked),
with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM
EDTA. The products of the reactions were visualized by the use of
an Hitachi FMBIO fluorescence imager. The results are displayed in
FIG. 35.
[0535] In FIG. 35, lane 1 contains the products of the reaction
containing the probe (SEQ ID NO:43), the invader oligonucleotide
(SEQ ID NO:46) and human genomic DNA. Examination of lane 1 shows
that the probe and invader oligonucleotides are specific for the
target sequence, and that the presence of genomic DNA does not
cause any significant background cleavage.
[0536] In FIG. 35, lanes 2 and 3 contain reaction products from
reactions containing the single-stranded target DNA (M13mp18), the
probe (SEQ ID NO:43) and the invader oligonucleotide (SEQ ID NO:46)
in the absence or presence of human genomic DNA, respectively.
Examination of lanes 2 and 3 demonstrate that the invader detection
assay may be used to detect the presence of a specific sequence on
a single-stranded target molecule in the presence or absence of a
large excess of competitor DNA (human genomic DNA).
[0537] In FIG. 35, lanes 4 and 5 contain reaction products from
reactions containing the double-stranded target DNA (M13mp19), the
probe (SEQ ID NO:43) and the invader oligonucleotide (SEQ ID NO:46)
in the absence or presence of human genomic DNA, respectively.
Examination of lanes 4 and 5 show that double stranded target
molecules are eminently suitable for invader-directed detection
reactions. The success of this reaction using a short duplexed
molecule, M13mp19, as the target in a background of a large excess
of genomic DNA is especially noteworthy as it would be anticipated
that the shorter and less complex M13 DNA strands would be expected
to find their complementary strand more easily than would the
strands of the more complex human genomic DNA. If the M13 DNA
reannealed before the probe and/or invader oligonucleotides could
bind to the target sequences along the M13 DNA, the cleavage
reaction would be prevented. In addition, because the denatured
genomic DNA would potentially contain regions complementary to the
probe and/or invader oligonucleotides it was possible that the
presence of the genomic DNA would inhibit the reaction by binding
these oligonucleotides thereby preventing their hybridization to
the M13 target. The above results demonstrate that these
theoretical concerns are not a problem under the reaction
conditions employed above.
[0538] In addition to demonstrating that the invader detection
assay may be used to detect sequences present in a double-stranded
target, these data also show that the presence of a large amount of
non-target DNA (470 ng/20 .mu.l reaction) does not lessen the
specificity of the cleavage. While this amount of DNA does show
some impact on the rate of product accumulation, probably by
binding a portion of the enzyme, the nature of the target sequence,
whether single- or double-stranded nucleic acid, does not limit the
application of this assay.
Example 15
Signal Accumulation in the Invader-Directed Cleavage Assay as a
Function of Target Concentration
[0539] To investigate whether the invader-directed cleavage assay
could be used to indicate the amount of target nucleic acid in a
sample, the following experiment was performed. Cleavage reactions
were assembled which contained an invader oligonucleotide (SEQ ID
NO:46), a labelled probe (SEQ ID NO:43) and a target nucleic acid,
M13 mp19. A series of reactions, which contained smaller and
smaller amounts of the M13 target DNA, was employed in order to
examine whether the cleavage products would accumulate in a manner
that reflected the amount of target DNA present in the
reaction.
[0540] The reactions were conducted as follows. A master mix
containing enzyme and buffer was assembled. Each 5 .mu.l of the
master mixture contained 25 ng of Cleavase.RTM. BN nuclease in 20
mM MOPS (pH 7.5) with 0.1% each of Tween 20 and NP-40, 4 mM
MnCl.sub.2 and 100 mM KCl. For each of the cleavage reactions shown
in lanes 4-13 of FIG. 36, a DNA mixture was generated which
contained 5 pmoles of the fluorescein-labelled probe
oligonucleotide (SEQ ID NO:43), 50 pmoles of the invader
oligonucleotide (SEQ ID NO:46) and 100, 50, 10, 5, 1, 0.5, 0.1,
0.05, 0.01 or 0.005 fmoles of single-stranded M13mp19,
respectively, for every 5 .mu.l of the DNA mixture. The DNA
solutions were covered with a drop of ChillOut.TM. evaporation
barrier (MJ Research) and brought to 61.degree. C. The cleavage
reactions were started by the addition of 5 .mu.l of the enzyme
mixture to each of tubes (final reaction volume was 10 .mu.l).
After 30 minutes at 61.degree. C., the reactions were terminated by
the addition of 8 .mu.l of 95% formamide with 20 mM EDTA and 0.05%
marker dyes. Samples were heated to 90.degree. C. for 1 minutes
immediately before electrophoresis through a 20% denaturing
acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer
containing 45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. To provide
reference (i.e., standards), 1.0, 0.1 and 0.01 pmole aliqouts of
fluorescein-labelled probe oligonucleotide (SEQ ID NO:43) were
diluted with the above formamide solution to a final volume of 18
.mu.l. These reference markers were loaded into lanes 1-3,
respectively of the gel. The products of the cleavage reactions (as
well as the reference standards) were visualized following
electrophoresis by the use of a Hitachi FMBIO fluorescence imager.
The results are displayed in FIG. 36.
[0541] In FIG. 36, boxes appear around fluorescein-containing
nucleic acid (i.e., the cleaved and uncleaved probe molecules) and
the amount of fluorescein contained within each box is indicated
under the box. The background fluorescence of the gel (see box
labelled "background") was subtracted by the fluoro-imager to
generate each value displayed under a box containing cleaved or
uncleaved probe products (the boxes are numbered 1-14 at top left
with a V followed by a number below the box). The lane marked "M"
contains fluoresceinated oligonucleotides which served as
markers.
[0542] The results shown in FIG. 36, demonstrate that the
accumulation of cleaved probe molecules in a fixed-length
incubation period reflects the amount of target DNA present in the
reaction. The results also demonstrate that the cleaved probe
products accumulate in excess of the copy number of the target.
This is clearly demonstrated by comparing the results shown in lane
3, in which 10 fmole (0.01 pmole) of uncut probe are displayed with
the results shown in 5, where the products which accumulated in
response to the presence of 10 fmole of target DNA are displayed.
These results show that the reaction can cleave hundreds of probe
oligonucleotide molecules for each target molecule present,
dramatically amplifying the target-specific signal generated in the
invader-directed cleavage reaction.
Example 16
Effect of Saliva Extract on the Invader-Directed Cleavage Assay
[0543] For a nucleic acid detection method to be useful in a
medical (i.e., a diagnostic) setting, it must not be inhibited by
materials and contaminants likely to be found in a typical clinical
specimen. To test the susceptibility of the invader-directed
cleavage assay to various materials, including but not limited to
nucleic acids, glycoproteins and carbohydrates, likely to be found
in a clinical sample, a sample of human saliva was prepared in a
manner consistent with practices in the clinical laboratory and the
resulting saliva extract was added to the invader-directed cleavage
assay. The effect of the saliva extract upon the inhibition of
cleavage and upon the specificity of the cleavage reaction was
examined.
[0544] One and one-half milliliters of human saliva were collected
and extracted once with an equal volume of a mixture containing
phenol:chloroform:isoamyl alcohol (25:24:1). The resulting mixture
was centrifuged in a microcentrifuge to separate the aqueous and
organic phases. The upper, aqueous phase was transferred to a fresh
tube. One-tenth volumes of 3 M NaOAc were added and the contents of
the tube were mixed. Two volumes of 100% ethyl alcohol were added
to the mixture and the sample was mixed and incubated at room
temperature for 15 minutes to allow a precipitate to form. The
sample was centrifuged in a microcentrifuge at 13,000 rpm for 5
minutes and the supernatant was removed and discarded. A milky
pellet was easily visible. The pellet was rinsed once with 70%
ethanol, dried under vacuum and dissolved in 200 .mu.l of 10 mM
Tris-HCl, pH 8.0, 0.1 mM EDTA (this constitutes the saliva
extract). Each .mu.l of the saliva extract was equivalent to 7.5
.mu.l of saliva. Analysis of the saliva extract by scanning
ultraviolet spectrophotometry showed a peak absorbance at about 260
nm and indicated the presence of approximately 45 ng of total
nucleic acid per .mu.l of extract.
[0545] The effect of the presence of saliva extract upon the
following enzymes was examined: Cleavase.RTM. BN nuclease,
Cleavase.RTM. A/G nuclease and three different lots of DNAPTaq:
AmpliTaq.RTM. (Perkin Elmer; a recombinant form of DNAPTaq),
AmpliTaq.RTM. LD (Perkin-Elmer; a recombinant DNAPTaq preparation
containing very low levels of DNA) and Taq DNA polymerase
(Fischer). For each enzyme tested, an enzyme/probe mixture was made
comprising the chosen amount of enzyme with 5 pmole of the probe
oligonucleotide (SEQ ID NO:43) in 10 .mu.l of 20 mM MOPS (pH 7.5)
containing 0.1% each of Tween 20 and NP-40, 4 mM MnCl.sub.2, 100 mM
KCl and 100 .mu.g/ml BSA. The following amounts of enzyme were
used: 25 ng of Cleavase.RTM. BN prepared as described in Example 9;
2 .mu.l of Cleavase.RTM. A/G nuclease extract prepared as described
in Example 2; 2.25 .mu.l (11.25 polymerase units) the following DNA
polymerases: AmpliTaq.RTM. DNA polymerase (Perkin Elmer);
AmpliTaq.RTM. DNA polymerase LD (low DNA; from Perkin Elmer); Taq
DNA polymerase (Fisher Scientific).
[0546] For each of the reactions shown in FIG. 37, except for that
shown in lane 1, the target DNA (50 fmoles of single-stranded
M13mp19 DNA) was combined with 50 pmole of the invader
oligonucleotide (SEQ ID NO:46) and 5 pmole of the probe
oligonucleotide (SEQ ID NO:43); target DNA was omitted in reaction
1 (lane 1). Reactions 1, 3, 5, 7, 9 and 11 included 1.5 .mu.l of
saliva extract. These mixtures were brought to a volume of 5 .mu.l
with distilled water, overlaid with a drop of ChillOut.RTM.
evaporation barrier (MJ Research) and brought to 95.degree. C. for
10 minutes. The cleavage reactions were then started by the
addition of 5 .mu.l of the desired enzyme/probe mixture; reactions
1, 4 and 5 received Cleavase.RTM. A/G nuclease. Reactions 2 and 3
received Cleavase.RTM. BN; reactions 6 and 7 received
AmpliTaq.RTM.; reactions 8 and 9 received AmoliTaq.RTM. LD; and
reactions 10 and 11 received Taq DNA Polymerase from Fisher
Scientific.
[0547] The reactions were incubated at 63.degree. C. for 30 minutes
and were stopped by the addition of 6 .mu.l of 95% formamide with
20 mM EDTA and 0.05% marker dyes. Samples were heated to 75.degree.
C. for 2 minutes immediately before electrophoresis through a 20%
acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of
45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The products of the
reactions were visualized by the use of an Hitachi FMBIO
fluorescence imager, and the results are displayed in FIG. 37.
[0548] A pairwise comparison of the lanes shown in FIG. 37 without
and with the saliva extract, treated with each of the enzymes,
shows that the saliva extract has different effects on each of the
enzymes. While the Cleavase.RTM. BN nuclease and the AmpliTaq.RTM.
are significantly inhibited from cleaving in these conditions, the
Cleavase.RTM. A/G nuclease and AmpliTaq.RTM. LD display little
difference in the yield of cleaved probe. The preparation of Taq
DNA polymerase from Fisher Scientific shows an intermediate
response, with a partial reduction in the yield of cleaved product.
From the standpoint of polymerization, the three DNAPTaq variants
should be equivalent; these should be the same protein with the
same amount of synthetic activity. It is possible that the
differences observed could be due to variations in the amount of
nuclease activity present in each preparation caused by different
handling during purification, or by different purification
protocols. In any case, quality control assays designed to assess
polymerization activity in commercial DNAP preparations would be
unlikely to reveal variation in the amount of nuclease activity
present. If preparations of DNAPTaq were screened for full 5'
nuclease activity (i.e., f the 5' nuclease activity was
specifically quantitated), it is likely that the preparations would
display sensitivities (to saliva extract) more in line with that
observed using Cleavase.RTM. A/G nuclease, from which DNAPTaq
differs by a very few amino acids.
[0549] It is worthy of note that even in the slowed reactions of
Cleavase.RTM. BN and the DNAPTaq variants there is no noticeable
increase in non-specific cleavage of the probe oligonucleotide due
to inappropriate hybridization or saliva-borne nucleases.
Example 17
[0550] Comparison of Additional 5' Nucleases in the
Invader-Directed Cleavage Assay
[0551] A number of eubacterial Type A DNA polymerases (i.e., Pol I
type DNA polymerases) have been shown to function as structure
specific endonucleases (Example 1 and Lyamichev et al., supra). In
this example, it was demonstrated that the enzymes of this class
can also be made to catalyze the invader-directed cleavage of the
present invention, albeit not as efficiently as the Cleavase.RTM.
enzymes.
[0552] Cleavase.RTM. BN nuclease and Cleavase.RTM. A/G nuclease
were tested along side three different thermostable DNA
polymerases: Thermus aquaticus DNA polymerase (Promega), Thermus
thermophilus and Thermus flavus DNA polymerases (Epicentre). The
enzyme mixtures used in the reactions shown in lanes 1-11 of FIG.
38 contained the following, each in a volume of 5 .mu.l: Lane 1: 20
mM MOPS (pH 7.5) with 0.1% each of Tween 20 and NP-40, 4 mM
MnCl.sub.2, 100 mM KCl; Lane 2: 25 ng of Cleavase.RTM. BN nuclease
in the same solution described for lane 1; Lane 3: 2.25 .mu.l of
Cleavase.RTM. A/G nuclease extract (prepared as described in
Example 2), in the same solution described for lane 1; Lane 4: 2.25
.mu.l of Cleavase.RTM. A/G nuclease extract in 20 mM Tris-Cl, (pH
8.5), 4 mM MgCl.sub.2 and 100 mM KCl; Lane 5: 11.25 polymerase
units of Taq DNA polymerase in the same buffer described for lane
4; Lane 6: 11.25 polymerase units of Tth DNA polymerase in the same
buffer described for lane 1; Lane 7: 11.25 polymerase units of Tth
DNA polymerase in a 2.times. concentration of the buffer supplied
by the manufacturer, supplemented with 4 mM MnCl.sub.2; Lane 8:
11.25 polymerase units of Tth DNA polymerase in a 2.times.
concentration of the buffer supplied by the manufacturer,
supplemented with 4 mM MgCl.sub.2; Lane 9: 2.25 polymerase units of
Tfl DNA polymerase in the same buffer described for lane 1; Lane
10: 2.25 polymerase units of Tfl polymerase in a 2.times.
concentration of the buffer supplied by the manufacturer,
supplemented with 4 mM MnCl.sub.2; Lane 11: 2.25 polymerase units
of Tfl DNA polymerase in a 2.times. concentration of the buffer
supplied by the manufacturer, supplemented with 4 mM
MgCl.sub.2.
[0553] Sufficient target DNA, probe and invader for all 11
reactions was combined into a master mix. This mix contained 550
fmoles of single-stranded M13mp19 target DNA, 550 pmoles of the
invader oligonucleotide (SEQ ID NO:46) and 55 pmoles of the probe
oligonucleotide (SEQ ID NO:43), each as depicted in FIG. 32c, in 55
.mu.l of distilled water. Five .mu.l of the DNA mixture was
dispensed into each of 11 labeled tubes and overlaid with a drop of
ChillOut.RTM. evaporation barrier (MJ Research). The reactions were
brought to 63.degree. C. and cleavage was started by the addition
of 5 .mu.l of the appropriate enzyme mixture. The reaction mixtures
were then incubated at 63.degree. C. temperature for 15 minutes.
The reactions were stopped by the addition of 8 .mu.l of 95%
formamide with 20 mM EDTA and 0.05% marker dyes. Samples were
heated to 90.degree. C. for 1 minute immediately before
electrophoresis through a 20% acrylamide gel (19:1 cross-linked),
with 7 M urea, in a buffer of 45 mM Tris-Borate (pH 8.3), 1.4 mM
EDTA. Following electrophoresis, the products of the reactions were
visualized by the use of an Hitachi FMBIO fluorescence imager, and
the results are displayed in FIG. 38. Examination of the results
shown in FIG. 38 demonstrates that all of the 5' nucleases tested
have the ability to catalyze invader-directed cleavage in at least
one of the buffer systems tested. Although not optimized here,
these cleavage agents are suitable for use in the methods of the
present invention.
Example 18
The Invader-Directed Cleavage Assay can Detect Single Base
Differences in Target Nucleic Acid Sequences
[0554] The ability of the invader-directed cleavage assay to detect
single base mismatch mutations was examined. Two target nucleic
acid sequences containing Cleavase.RTM. enzyme-resistant
phosphorothioate backbones were chemically synthesized and purified
by polyacrylamide gel electrophoresis. Targets comprising
phosphorothioate backbones were used to prevent exonucleolytic
nibbling of the target when duplexed with an oligonucleotide. A
target oligonucleotide, which provides a target sequence that is
completely complementary to the invader oligonucleotide (SEQ ID
NO:46) and the probe oligonucleotide (SEQ ID NO:43), contained the
following sequence:
5'-CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC-3' (SEQ ID NO:47). A
second target sequence containing a single base change relative to
SEQ ID NO:47 was synthesized: 5'-CCTTTCGCTCTCTTCCCTTCCTTTCTCGCC
ACGTTCGCCGGC-3 (SEQ ID NO:48; the single base change relative to
SEQ. ID NO:47 is shown using bold and underlined type). The
consequent mismatch occurs within the "Z" region of the target as
represented in FIG. 29.
[0555] To discriminate between two target sequences which differ by
the presence of a single mismatch), invader-directed cleavage
reactions were conducted using two different reaction temperatures
(55.degree. C. and 60.degree. C.). Mixtures containing 200 fmoles
of either SEQ ID NO:47 or SEQ ID NO:48, 3 pmoles of
fluorescein-labelled probe oligonucleotide (SEQ ID NO:43), 7.7
pmoles of invader oligonucleotide (SEQ ID NO:46) and 2 ill of
Cleavase.RTM. A/G nuclease extract (prepared as described in
Example 2) in 9 .mu.l of 10 mM MOPS (pH 7.4) with 50 mM KCl were
assembled, covered with a drop of ChillOut.RTM. evaporation barrier
(MJ Research) and brought to the appropriate reaction temperature.
The cleavage reactions were initiated by the addition of 1 .mu.l of
20 mM MgCl.sub.2. After 30 minutes at either 55.degree. C. or
60.degree. C., 10 ill of 95% formamide with 20 mM EDTA and 0.05%
marker dyes was added to stop the reactions. The reaction mixtures
where then heated to 90.degree. C. for one minute prior to loading
4 .mu.l onto 20% denaturing polyacrylamide gels. The resolved
reaction products were visualized using a Hitachi FMBIO
fluorescence imager. The resulting image is shown in FIG. 39.
[0556] In FIG. 39, lanes 1 and 2 show the products from reactions
conducted at 55.degree. C.; lanes 3 and 4 show the products from
reactions conducted at 60.degree. C. Lanes 1 and 3 contained
products from reactions containing SEQ ID NO:47 (perfect match to
probe) as the target. Lanes 2 and 4 contained products from
reactions containing SEQ ID NO:48 (single base mis-match with
probe) as the target. The target that does not have a perfect
hybridization match (i.e., complete complementarity) with the probe
will not bind as strongly, i.e., the T.sub.m of that duplex will be
lower than the T.sub.m of the same region if perfectly matched. The
results presented here show that reaction conditions can be varied
to either accommodate the mis-match (e.g., by lowering the
temperature of the reaction) or to exclude the binding of the
mis-matched sequence (e.g., by raising the reaction
temperature).
[0557] The results shown in FIG. 39 demonstrate that the specific
cleavage event which occurs in invader-directed cleavage reactions
can be eliminated by the presence of a single base mis-match
between the probe oligonucleotide and the target sequence. Thus,
reaction conditions can be chosen so as to exclude the
hybridization of mis-matched invader-directed cleavage probes
thereby diminishing or even eliminating the cleavage of the probe.
In an extension of this assay system, multiple cleavage probes,
each possessing a separate reporter molecule (i.e., a unique
label), could also be used in a single cleavage reaction, to
simultaneously probe for two or more variants in the same target
region. The products of such a reaction would allow not only the
detection of mutations which exist within a target molecule, but
would also allow a determination of the relative concentrations of
each sequence (i.e., mutant and wild type or multiple different
mutants) present within samples containing a mixture of target
sequences. When provided in equal amounts, but in a vast excess
(e.g. at least a 100-fold molar excess; typically at least 1 pmole
of each probe oligonucleotide would be used when the target
sequence was present at about 10 fmoles or less) over the target
and used in optimized conditions. As discussed above, any
differences in the relative amounts of the target variants will not
affect the kinetics of hybridization, so the amounts of cleavage of
each probe will reflect the relative amounts of each variant
present in the reaction.
[0558] The results shown in the example clearly demonstrate that
the invader-directed cleavage reaction can be used to detect single
base difference between target nucleic acids.
Example 19
The Invader-Directed Cleavage Reaction is Insensitive to Large
Changes in Reaction Conditions
[0559] The results shown above demonstrated that the
invader-directed cleavage reaction can be used for the detection of
target nucleic acid sequences and that this assay can be used to
detect single base difference between target nucleic acids. These
results demonstrated that 5' nucleases (e.g., Cleavase.RTM.BN,
Cleavase.RTM. A/G, DNAPTaq, DNAPTth, DNAPTf1) could be used in
conjunction with a pair of overlapping oligonucleotides as an
efficient way to recognize nucleic acid targets. In the experiments
below it is demonstrated that invasive cleavage reaction is
relatively insensitive to large changes in conditions thereby
making the method suitable for practice in clinical
laboratories.
[0560] The effects of varying the conditions of the cleavage
reaction were examined for their effect(s) on the specificity of
the invasive cleavage and the on the amount of signal accumulated
in the course of the reaction. To compare variations in the
cleavage reaction a "standard" invader cleavage reaction was first
defined. In each instance, unless specifically stated to be
otherwise, the indicated parameter of the reaction was varied,
while the invariant aspects of a particular test were those of this
standard reaction. The results of these tests are shown in FIGS.
42-51.
[0561] a) The Standard Invader-Directed Cleavage Reaction
[0562] The standard reaction was defined as comprising 1 fmole of
M13mp18 single-stranded target DNA (New England Biolabs), 5 pmoles
of the labeled probe oligonucleotide (SEQ ID NO:49), 10 pmole of
the upstream invader oligonucleotide (SEQ ID NO:50) and 2 units of
Cleavase.RTM. A/G in 10 .mu.l of 10 mM MOPS, pH 7.5 with 100 mM
KCl, 4 mM MnCl.sub.2, and 0.05% each Tween-20 and Nonidet-P40. For
each reaction, the buffers, salts and enzyme were combined in a
volume of 5 .mu.l; the DNAs (target and two oligonucleotides) were
combined in 5 .mu.l of dH2O and overlaid with a drop of
ChillOut.RTM. evaporation barrier (MJ Research). When multiple
reactions were performed with the same reaction constituents, these
formulations were expanded proportionally.
[0563] Unless otherwise stated, the sample tubes with the DNA
mixtures were warmed to 61.degree. C., and the reactions were
started by the addition of 5 .mu.l of the enzyme mixture. After 20
minutes at this temperature, the reactions were stopped by the
addition of 8 .mu.l of 95% formamide with 20 mM EDTA and 0.05%
marker dyes. Samples were heated to 75.degree. C. for 2 minutes
immediately before electrophoresis through a 20% acrylamide gel
(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM
Tris-Borate, pH 8.3, 1.4 mM EDTA. The products of the reactions
were visualized by the use of an Hitachi FMBIO fluorescence imager.
In each case, the uncut probe material was visible as an intense
black band or blob, usually in the top half of the panel, while the
desired products of invader specific cleavage were visible as one
or two narrower black bands, usually in the bottom half of the
panel. Under some reaction conditions, particulary those with
elevated salt concentrations, a secondary cleavage product is also
visible (thus generating a doublet). Ladders of lighter grey bands
generally indicate either exonuclease nibbling of the probe
oligonucleotide or heat-induced, non-specific breakage of the
probe.
[0564] FIG. 41 depicts the annealing of the probe and invader
oligonucleotides to regions along the M13mp18 target molecule (the
bottom strand). In FIG. 41 only a 52 nucleotide portion of the M13
mp18 molecule is shown; this 52 nucleotide sequence is listed in
SEQ ID NO:42 (this sequence is identical in both M13 mp18 and M13
mp19). The probe oligonucleotide (top strand) contains a Cy3
amidite label at the 5' end; the sequence of the probe is
5'-AGAAAGGAAGGGAAGAAAGCGAAA GGT-3' (SEQ ID NO:49. The bold type
indicates the presence of a modified base (2'-O--CH.sub.3). Cy3
amidite (Pharmacia) is a indodicarbocyanine dye amidite which can
be incorporated at any position during the synthesis of
oligonucleotides; Cy3 fluoresces in the yellow region (excitation
and emission maximum of 554 and 568 nm, respectively). The invader
oligonucleotide (middle strand) has the following sequence:
TABLE-US-00002 5'-GCCGGCGAACGTGGCGAGAAAGGA-3'. (SEQ ID NO:50)
[0565] b) KCl Titration
[0566] FIG. 42 shows the results of varying the KCl concentration
in combination with the use of 2 mM MnCl.sub.2, in an otherwise
standard reaction. The reactions were performed in duplicate for
confirmation of observations; the reactions shown in lanes 1 and 2
contained no added KCl, lanes 3 and 4 contained KCl at 5 mM, lanes
5 and 6 contained 25 mM KCl, lanes 7 and 8 contained 50 mM KCl,
lanes 9 and 10 contained 100 mM KCl and lanes 11 and 12 contained
200 mM KCl. These results show that the inclusion of KCl allows the
generation of a specific cleavage product. While the strongest
signal is observed at the 100 mM KCl concentration, the specificity
of signal in the other reactions with KCl at or above 25 mM
indicates that concentrations in the full range (i.e., 25-200 mM)
may be chosen if it is so desirable for any particular reaction
conditions.
[0567] As shown in FIG. 42, the invader-directed cleavage reaction
requires the presence of salt (e.g., KCl) for effective cleavage to
occur. In other reactions, it has been found that KCl can inhibit
the activity of certain Cleavase.RTM. enzymes when present at
concentrations above about 25 mM (For example, in cleavage
reactions using the S-60 oligonucleotide shown in FIG. 30, in the
absence of primer, the Cleavase.RTM. BN enzyme loses approximately
50% of its activity in 50 mM KCl). Therefore, the use of
alternative salts in the invader-directed cleavage reaction was
examined. In these experiments, the potassium ion was replaced with
either Na.sup.+ or Li.sup.+ or the chloride ion was replaced with
glutamic acid. The replacement of KCl with alternative salts is
described below in sections c-e.
c) NaCl Titration
[0568] FIG. 43 shows the results of using various concentrations of
NaCl in place of KCl (lanes 3-10) in combination with the use 2 mM
MnCl.sub.2, in an otherwise standard reaction, in comparison to the
effects seen with 100 mM KCl (lanes 1 and 2). The reactions
analyzed in lanes 3 and 4 contained NaCl at 75 mM, lanes 5 and 6
contained 100 mM, lanes 7 and 8 contained 150 mM and lanes 9 and 10
contained 200 mM. These results show that NaCl can be used as a
replacement for KCl in the invader-directed cleavage reaction
(i.e., the presence of NaCl, like KCl, enhances product
accumulation).
[0569] d) LiCl Titration
[0570] FIG. 44 shows the results of using various concentrations of
LiCl in place of KCl (lanes 3-14) in otherwise standard reactions,
compared to the effects seen with 100 mM KCl (lanes 1 and 2). The
reactions analyzed in lanes 3 and 4 contained LiCl at 25 mM, lanes
5 and 6 contained 50 mM, lanes 7 and 8 contained 75 mM, lanes 9 and
10 contained 100 mM, lanes 11 and 12 contained 150 mM and lanes 13
and 14 contained 200 mM. These results demonstrate that LiCl can be
used as a suitable replacement for KCl in the invader-directed
cleavage reaction (i.e., the presence of LiCl, like KCl, enhances
product accumulation).
[0571] e) KGlu Titration
[0572] FIG. 45 shows the results of using a glutamate salt of
potassium (KGlu) in place of the more commonly used chloride salt
(KCl) in reactions performed over a range of temperatures. KGlu has
been shown to be a highly effective salt source for some enzymatic
reactions, showing a broader range of concentrations which permit
maximum enzymatic activity [Leirmo et al. (1987) Biochem. 26:2095].
The ability of KGlu to facilitate the annealing of the probe and
invader oligonucleotides to the target nucleic acid was compared to
that of LiCl. In these experiments, the reactions were run for 15
minutes, rather than the standard 20 minutes. The reaction analyzed
in lane 1 contained 150 mM LiCl and was run at 65.degree. C.; the
reactions analyzed in lanes 2-4 contained 200 mM, 300 mM and 400 mM
KGlu, respectively and were run at 65.degree. C. The reactions
analyzed in lanes 5-8 repeated the array of salt concentrations
used in lanes 1-4, but were performed at 67.degree. C.; lanes 9-12
show the same array run at 69.degree. C. and lanes 13-16 show the
same array run at 71.degree. C. The results shown in FIG. 45
demonstrate that KGlu was very effective as a salt in the invasive
cleavage reactions. In addition, these data show that the range of
allowable KGlu concentrations was much greater than that of LiCl,
with full activity apparent even at 400 mM KGlu.
[0573] f) MnCl.sub.2 and MgCl.sub.2 Titration and Ability to
Replace MnCl.sub.2 With MgCl.sub.2
[0574] In some instances it may be desirable to perform the
invasive cleavage reaction in the presence of Mg.sup.2+, either in
addition to, or in place of Mn.sup.2+ as the necessary divalent
cation required for activity of the enzyme employed. For example,
some common methods of preparing DNA from bacterial cultures or
tissues use MgCl.sub.2 in solutions which are used to facilitate
the collection of DNA by precipitation. In addition, elevated
concentrations (i.e., greater than 5 mM) of divalent cation can be
used to facilitate hybridization of nucleic acids, in the same way
that the monovalent salts were used above, thereby enhancing the
invasive cleavage reaction. In this experiment, the tolerance of
the invasive cleavage reaction was examined for 1) the substitution
of MgCl.sub.2 for MnCl.sub.2 and for the ability to produce
specific product in the presence of increasing concentrations of
MgCl.sub.2 and MnCl.sub.2.
[0575] FIG. 46 shows the results of either varying the
concentration of MnCl.sub.2 from 2 mM to 8 mM, replacing the
MnCl.sub.2 with MgCl.sub.2 at 2 to 4 mM, or of using these
components in combination in an otherwise standard reaction. The
reactions analyzed in lanes 1 and 2 contained 2 mM each MnCl.sub.2
and MgCl.sub.2, lanes 3 and 4 contained 2 mM MnCl.sub.2 only, lanes
5 and 6 contained 3 mM MnCl.sub.2, lanes 7 and 8 contained 4 mM
MnCl.sub.2, lanes 9 and 10 contained 8 mM MnCl.sub.2. The reactions
analyzed in lanes 11 and 12 contained 2 mM MgCl.sub.2 and lanes 13
and 14 contained 4 mM MgCl.sub.2. These results show that both
MnCl.sub.2 and MgCl.sub.2 can be used as the necessary divalent
cation to enable the cleavage activity of the Cleavase.RTM. A/G
enzyme in these reactions and that the invasive cleavage reaction
can tolerate a broad range of concentrations of these
components.
[0576] In addition to examining the effects of the salt environment
on the rate of product accumulation in the invasive cleavage
reaction, the use of reaction constituents shown to be effective in
enhancing nucleic acid hybridization in either standard
hybridization assays (e.g., blot hybridization) or in ligation
reactions was examined. These components may act as volume
excluders, increasing the effective concentration of the nucleic
acids of interest and thereby enhancing hybridization, or they may
act as charge-shielding agents to minimize repulsion between the
highly charged backbones of the nucleic acids strands. The results
of these experiments are described in sections g and h below.
[0577] g) Effect of CTAB Addition
[0578] The polycationic detergent cetyltrietheylammonium bromide
(CTAB) has been shown to dramatically enhance hybridization of
nucleic acids [Pontius and Berg (1991) Proc. Natl. Acad. Sci. USA
88:8237]. The data shown in FIG. 47 depicts the results of adding
the detergent CTAB to invasive cleavage reactions in which 150 mM
LiCl was used in place of the KCl in otherwise standard reactions.
Lane 1 shows unreacted (i.e., uncut) probe, and the reaction shown
in lane 1 is the LiCl modified standard reaction without CTAB. The
reactions analyzed in lanes 3 and 4 contained 100 .mu.M CTAB, lanes
5 and 6 contained 200 .mu.M CTAB, lanes 7 and 8 contained 400 .mu.M
CTAB, lanes 9 and 10 contained 600 .mu.M CTAB, lanes 11 and 12
contained 800 .mu.M CTAB and lanes 13 and 14 contained 1 mM CTAB.
These results showed that the lower amounts of CTAB may have a very
moderate enhancing effect under these reaction conditions, and the
presence of CTAB in excess of about 500 .mu.M was inhibitory to the
accumulation of specific cleavage product.
[0579] h) Effect Of PEG Addition
[0580] FIG. 48 shows the effect of adding polyethylene glycol (PEG)
at various percentage (w/v) concentrations to otherwise standard
reactions. The effects of increasing the reaction temperature of
the PEG-containing reactions was also examined. The reactions
assayed in lanes 1 and 2 were the standard conditions without PEG,
lanes 3 and 4 contained 4% PEG, lanes 5 and 6 contained 8% PEG and
lanes 7 and 8 contained 12% PEG. Each of the aforementioned
reactions was performed at 61.degree. C. The reactions analyzed in
lanes 9, 10, 11 and 12 were performed at 65.degree. C., and
contained 0%, 4%, 8% and 12% PEG, respectively. These results show
that at all percentages tested, and at both temperatures tested,
the inclusion of PEG substantially eliminated the production of
specific cleavage product.
[0581] In addition to the data presented above (i.e., effect of
CTAB and PEG addition), the presence of 1.times. Denhardts in the
reaction mixture was found to have no adverse effect upon the
cleavage reaction [50.times. Denhardt's contains per 500 ml: 5 g
Ficoll, 5 g polyvinylpyrrolidone, 5 g BSA]. In addition, the
presence of each component of Denhardt's was examined individually
(i.e., Ficoll alone, polyvinylpyrrolidone alone, BSA alone) for the
effect upon the invader-directed cleavage reaction; no adverse
effect was observed.
[0582] i) Effect of the Addition of Stabilizing Agents
[0583] Another approach to enhancing the output of the invasive
cleavage reaction is to enhance the activity of the enzyme
employed, either by increasing its stability in the reaction
environment or by increasing its turnover rate. Without regard to
the precise mechanism by which various agents operate in the
invasive cleavage reaction, a number of agents commonly used to
stabilize enzymes during prolonged storage were tested for the
ability to enhance the accumulation of specific cleavage product in
the invasive cleavage reaction.
[0584] FIG. 49 shows the effects of adding glycerol at 15% and of
adding the detergents Tween-20 and Nonidet-P40 at 1.5%, alone or in
combination, in otherwise standard reactions. The reaction analyzed
in lane 1 was a standard reaction. The reaction analyzed in lane 2
contained 1.5% NP-40, lane 3 contained 1.5% Tween 20, lane 4
contained 15% glycerol. The reaction analyzed in lane 5 contained
both Tween-20 and NP-40 added at the above concentrations, lane 6
contained both glycerol and NP-40, lane 7 contained both glycerol
and Tween-20, and lane 8 contained all three agents. The results
shown in FIG. 49 demonstrate that under these conditions these
adducts had little or no effect on the accumulation of specific
cleavage product.
[0585] FIG. 50 shows the effects of adding gelatin to reactions in
which the salt identity and concentration were varied from the
standard reaction. In addition, all of these reactions were
performed at 65.degree. C., instead of 61.degree. C. The reactions
assayed in lanes 1-4 lacked added KCl, and included 0.02%, 0.05%,
0.1% or 0.2% gelatin, respectively. Lanes 5, 6, 7 and 8 contained
the same titration of gelatin, respectively, and included 100 mM
KCl. Lanes 9, 10, 11 and 12, also had the same titration of
gelatin, and additionally included 150 mM LiCl in place of KCl.
Lanes 13 and 14 show reactions that did not include gelatin, but
which contained either 100 mM KCl or 150 mM LiCl, respectively. The
results shown in FIG. 50 demonstrated that in the absence of salt
the gelatin had a moderately enhancing effect on the accumulation
of specific cleavage product, but when either salt (KCl or LiCl)
was added to reactions performed under these conditions, increasing
amounts of gelatin reduced the product accumulation.
[0586] j) Effect of Adding Large Amounts of Non-Target Nucleic
Acid
[0587] In detecting specific nucleic acid sequences within samples,
it is important to determine if the presence of additional genetic
material (i.e., non-target nucleic acids) will have a negative
effect on the specificity of the assay. In this experiment, the
effect of including large amounts of non-target nucleic acid,
either DNA or RNA, on the specificity of the invasive cleavage
reaction was examined. The data was examined for either an
alteration in the expected site of cleavage, or for an increase in
the nonspecific degradation of the probe oligonucleotide.
[0588] FIG. 51 shows the effects of adding non-target nucleic acid
(e.g., genomic DNA or tRNA) to an invasive cleavage reaction
performed at 65.degree. C., with 150 mM LiCl in place of the KCl in
the standard reaction. The reactions assayed in lanes 1 and 2
contained 235 and 470 ng of genomic DNA, respectively. The
reactions analyzed in lanes 3, 4, 5 and 6 contained 100 ng, 200 ng,
500 ng and 1 .mu.g of tRNA, respectively. Lane 7 represents a
control reaction which contained no added nucleic acid beyond the
amounts used in the standard reaction. The results shown in FIG. 51
demonstrate that the inclusion of non-target nucleic acid in large
amounts could visibly slow the accumulation of specific cleavage
product (while not limiting the invention to any particular
mechanism, it is thought that the additional nucleic acid competes
for binding of the enzyme with the specific reaction components).
In additional experiments it was found that the effect of adding
large amounts of non-target nucleic acid can be compensated for by
increasing the enzyme in the reaction. The data shown in FIG. 51
also demonstrate that a key feature of the invasive cleavage
reaction, the specificity of the detection, was not compromised by
the presence of large amounts of non-target nucleic acid.
[0589] In addition to the data presented above, invasive cleavage
reactions were run with succinate buffer at pH 5.9 in place of the
MOPS buffer used in the "standard" reaction; no adverse effects
were observed.
[0590] The data shown in FIGS. 42-51 and described above
demonstrate that the invasive cleavage reaction can be performed
using a wide variety of reaction conditions and is therefore
suitable for practice in clinical laboratories.
Example 20
[0591] Detection of RNA Targets by Invader-Directed Cleavage
[0592] In addition to the clinical need to detect specific DNA
sequences for infectious and genetic diseases, there is a need for
technologies that can quantitatively detect target nucleic acids
that are composed of RNA. For example, a number of viral agents,
such as hepatitis C virus (HCV) and human immunodeficiency virus
(HIV) have RNA genomic material, the quantitative detection of
which can be used as a measure of viral load in a patient sample.
Such information can be of critical diagnostic or prognostic
value.
[0593] Hepatitis C virus (HCV) infection is the predominant cause
of post-transfusion non-A, non-B (NANB) hepatitis around the world.
In addition, HCV is the major etiologic agent of hepatocellular
carcinoma (HCC) and chronic liver disease world wide. The genome of
HCV is a small (9.4 kb) RNA molecule. In studies of transmission of
HCV by blood transfusion it has been found the presence of HCV
antibody, as measured in standard immunological tests, does not
always correlate with the infectivity of the sample, while the
presence of HCV RNA in a blood sample strongly correlates with
infectivity. Conversely, serological tests may remain negative in
immunosuppressed infected individuals, while HCV RNA may be easily
detected [J. A. Cuthbert (1994) Clin. Microbiol. Rev. 7:505].
[0594] The need for and the value of developing a probe-based assay
for the detection the HCV RNA is clear. The polymerase chain
reaction has been used to detect HCV in clinical samples, but the
problems associated with carry-over contamination of samples has
been a concern. Direct detection of the viral RNA without the need
to perform either reverse transcription or amplification would
allow the elimination of several of the points at which existing
assays may fail.
[0595] The genome of the positive-stranded RNA hepatitis C virus
comprises several regions including 5' and 3' noncoding regions
(i.e., 5' and 3' untranslated regions) and a polyprotein coding
region which encodes the core protein (C), two envelope
glycoproteins (E1 and E2/NS1) and six nonstructural glycoproteins
(NS2-NS5b). Molecular biological analysis of the HCV genome has
showed that some regions of the genome are very highly conserved
between isolates, while other regions are fairly rapidly
changeable. The 5' noncoding region (NCR) is the most highly
conserved region in the HCV. These analyses have allowed these
viruses to be divided into six basic genotype groups, and then
further classified into over a dozen sub-types [the nomenclature
and division of HCV genotypes is evolving; see Altamirano et al.,
J. Infect. Dis. 171:1034 (1995) for a recent classification
scheme].
[0596] In order to develop a rapid and accurate method of detecting
HCV present in infected individuals, the ability of the
invader-directed cleavage reaction to detect HCV RNA was examined.
Plasmids containing DNA derived from the conserved 5'-untranslated
region of six different HCV. RNA isolates were used to generate
templates for in vitro transcription. The HCV sequences contained
within these six plasmids represent genotypes 1 (four sub-types
represented; 1a, 1b, 1c, and .DELTA.1c), 2, and 3. The nomenclature
of the HCV genotypes used herein is that of Simmonds et al. [as
described in Altamirano et at., supra]. The Alc subtype was used in
the model detection reaction described below.
[0597] a) Generation of Plasmids Containing HCV Sequences
[0598] Six DNA fragments derived from HCV were generated by RT-PCR
using RNA extracted from serum samples of blood donors; these PCR
fragments were a gift of Dr. M. Altamirano (University of British
Columbia. Vancouver). These PCR fragments represent HCV sequences
derived from HCV genotypes 1a, 1b, 1c, .DELTA.1c, 2c and 3a.
[0599] The RNA extraction, reverse transcription and PCR were
performed using standard techniques (Altamirano et al., supra).
Briefly, RNA was extracted from 100 .mu.l of serum using guanidine
isothiocyanate, sodium lauryl sarkosate and phenol-chloroform
[Inchauspe et al., Hepatology 14:595 (1991)]. Reverse transcription
was performed according to the manufacturer's instructions using a
GeneAmp rTh reverse transcriptase RNA PCR kit (Perkin-Elmer) in the
presence of an external antisense primer, HCV342. The sequence of
the HCV342 primer is 5'-GGTTTTTCTTTGAGG TTTAG-3' (SEQ ID NO:51).
Following termination of the RT reaction, the sense primer HCV7
[5'-GCGACACTCCACCATAGAT-3' (SEQ ID NO:52)] and magnesium were added
and a first PCR was performed. Aliquots of the first PCR products
were used in a second (nested) PCR in the presence of primers HCV46
[5'-CTGTCTTCACGCAGAAAGC-3' (SEQ ID NO:53)] and HCV308 [5'-GCACGGT
CTACGAGACCTC-3' (SEQ ID NO:54)]. The PCRs produced a 281 bp product
which corresponds to a conserved 5' noncoding region (NCR) region
of HCV between positions -284 and -4 of the HCV genome (Altramirano
et al., supra).
[0600] The six 281 bp PCR fragments were used directly for cloning
or they were subjected to an additional amplification step using a
50 .mu.l PCR comprising approximately 100 fmoles of DNA, the HCV46
and HCV308 primers at 0.1 .mu.M, 100 .mu.M of all four dNTPs and
2.5 units of Taq DNA polymerase in a buffer containing 10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2 and 0.1% Tween 20.
The PCRs were cycled 25 times at 96.degree. C. for 45 sec.,
55.degree. C. for 45 sec. and 72.degree. C. for 1 min. Two
microliters of either the original DNA samples or the reamplified
PCR products were used for cloning in the linear pT7Blue T-vector
(Novagen, Madison, Wis.) according to manufacturer's protocol.
After the PCR products were ligated to the pT7Blue T-vector, the
ligation reaction mixture was used to transform competent JM109
cells (Promega). Clones containing the pT7Blue T-vector with an
insert were selected by the presence of colonies having a white
color on LB plates containing 40 .mu.g/ml X-Gal, 40 .mu.g/ml IPTG
and 50 .mu.g/ml ampicillin. Four colonies for each PCR sample were
picked and grown overnight in 2 ml LB media containing 50 .mu.g/ml
carbenicillin. Plasmid DNA was isolated using the following
alkaline miniprep protocol. Cells from 1.5 ml of the overnight
culture were collected by centrifugation for 2 min. in a
microcentrifuge (14K rpm), the supernatant was discarded and the
cell pellet was resuspended in 50 .mu.l TE buffer with 10 .mu.g/ml
RNAse A (Pharmacia). One hundred microliters of a solution
containing 0.2 N NaOH, 1% SDS was added and the cells were lysed
for 2 min. The lysate was gently mixed with 100 .mu.l of 1.32 M
potassium acetate, pH 4.8, and the mixture was centrifuged for 4
min. in a microcentrifuge (14K rpm); the pellet comprising cell
debris was discarded. Plasmid DNA was precipitated from the
supernatant with 200 .mu.l ethanol and pelleted by centrifugation a
microcentrifuge (14K rpm). The DNA pellet was air dried for 15 min.
and was then redissolved in 50 .mu.l TE buffer (10 mM Tris-HCl, pH
7.8, 1 mM EDTA).
[0601] b). Reamplification of HCV Clones to Add The Phage T7
Promoter for Subsequent in Vitro Transcription
[0602] To ensure that the RNA product of transcription had a
discrete 3' end it was necessary to create linear transcription
templates which stopped at the end of the HCV sequence. These
fragments were conveniently produced using the PCR to reamplify the
segment of the plasmid containing the phage promoter sequence and
the HCV insert. For these studies, the clone of HCV type .DELTA.1c
was reamplified using a primer that hybridizes to the T7 promoter
sequence: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO:55; "the T7
promoter primer") (Novagen) in combination with the 3' terminal
HCV-specific primer HCV308 (SEQ ID NO:54). For these reactions, 1
.mu.l of plasmid DNA (approximately 10 to 100 ng) was reamplified
in a 200 .mu.l PCR using the T7 and HCV308 primers as described
above with the exception that 30 cycles of amplification were
employed. The resulting amplicon was 354 bp in length. After
amplification the PCR mixture was transferred to a fresh 1.5 ml
microcentrifuge tube, the mixture was brought to a final
concentration of 2 M NH.sub.4OAc, and the products were
precipitated by the addition of one volume of 100% isopropanol.
Following a 10 min. incubation at room temperature, the
precipitates were collected by centrifugation, washed once with 80%
ethanol and dried under vacuum. The collected material was
dissolved in 100 .mu.l nuclease-free distilled water (Promega).
[0603] Segments of RNA were produced from this amplicon by in vitro
transcription using the RiboMAX.TM. Large Scale RNA Production
System (Promega) in accordance with the manufacturer's
instructions, using 5.3 .mu.g of the amplicon described above in a
100 .mu.l reaction. The transcription reaction was incubated for
3.75 hours, after which the DNA template was destroyed by the
addition of 5-6 .mu.l of RQ1 RNAse-free DNAse (1 unit/.mu.l)
according to the RiboMAX.TM. kit instructions. The reaction was
extracted twice with phenol/chloroform/isoamyl alcohol (50:48:2)
and the aqueous phase was transferred to a fresh microcentrifuge
tube. The RNA was then collected by the addition of 10 .mu.l of 3M
NH.sub.4OAc, pH 5.2 and 110 .mu.l of 100% isopropanol. Following a
5 min. incubation at 4.degree. C., the precipitate was collected by
centrifugation, washed once with 80% ethanol and dried under
vacuum. The sequence of the resulting RNA transcript (HCV1.1
transcript) is listed in SEQ ID NO:56.
[0604] c) Detection of the HCV1.1 Transcript in the
Invader-Directed Cleavage Assay
[0605] Detection of the HCV1.1 transcript was tested in the
invader-directed cleavage assay using an HCV-specific probe
oligonucleotide [5'-CCGGTCGTCCTGGCAAT XCC-3' (SEQ ID NO:57); X
indicates the presence of a fluorescein dye on an abasic linker)
and an HCV-specific invader oligonucleotide [5'-GTTTATCCAAGAAAGGAC
CCGGTCC-3' (SEQ ID NO:58)] that causes a 6-nucleotide invasive
cleavage of the probe.
[0606] Each 10 .mu.l of reaction mixture comprised 5 pmole of the
probe oligonucleotide (SEQ ID NO:57) and 10 pmole of the invader
oligonucleotide (SEQ ID NO:58) in a buffer of 10 mM MOPS, pH 7.5
with 50 mM KCl, 4 mM MnCl.sub.2, 0.05% each Tween-20 and
Nonidet-P40 and 7.8 units RNasine ribonuclease inhibitor (Promega).
The cleavage agents employed were Cleavase.RTM. A/G (used at 5.3
ng/10 .mu.l reaction) or DNAPTth (used at 5 polymerase units/10
.mu.l reaction). The amount of RNA target was varied as indicated
below. When RNAse treatment is indicated, the target RNAs were
pre-treated with 10 .mu.g of RNase A (Sigma) at 37.degree. C. for
30 min. to demonstrate that the detection was specific for the RNA
in the reaction and not due to the presence of any residual DNA
template from the transcription reaction. RNase-treated aliquots of
the HCV RNA were used directly without intervening
purification.
[0607] For each reaction, the target RNAs were suspended in the
reaction solutions as described above, but lacking the cleavage
agent and the MnCl.sub.2 for a final volume of 10 .mu.l, with the
invader and probe at the concentrations listed above. The reactions
were warmed to 46.degree. C. and the reactions were started by the
addition of a mixture of the appropriate enzyme with MnCl.sub.2.
After incubation for 30 min. at 46.degree. C., the reactions were
stopped by the addition of 8 .mu.l of 95% formamide, 10 mM EDTA and
0.02% methyl violet (methyl violet loading buffer). Samples were
then resolved by electrophoresis through a 15% denaturing
polyacrylamide gel (19:1 cross-linked), containing 7 M urea, in a
buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Following
electrophoresis, the labeled reaction products were visualized
using the FMBIO-100 Image Analyzer (Hitachi), with the resulting
imager scan shown in FIG. 52.
[0608] In FIG. 52, the samples analyzed in lanes 1-4 contained 1
pmole of the RNA target, the reactions shown in lanes 5-8 contained
100 fmoles of the RNA target and the reactions shown in lanes 9-12
contained 10 fmoles of the RNA target. All odd-numbered lanes
depict reactions performed using Cleavase.RTM. A/G enzyme and all
even-numbered lanes depict reactions performed using DNAPTth. The
reactions analyzed in lanes 1, 2, 5, 6, 9 and 10 contained RNA that
had been pre-digested with RNase A. These data demonstrate that the
invasive cleavage reaction efficiently detects RNA targets and
further, the absence of any specific cleavage signal in the
RNase-treated samples confirms that the specific cleavage product
seen in the other lanes is dependent upon the presence of input
RNA.
Example 21
The Fate of the Target RNA in the Invader-Directed Cleavage
Reaction
[0609] In this example, the fate of the RNA target in the
invader-directed cleavage reaction was examined. As shown above in
Example 1D, when RNAs are hybridized to DNA oligonucleotides, the
5' nucleases associated with DNA polymerases can be used to cleave
the RNAs; such cleavage can be suppressed when the 5' arm is long
or when it is highly structured [Lyamichev et al. (1993) Science
260:778 and U.S. Pat. No. 5,422,253, the disclosure of which is
herein incorporated by reference]. In this experiment, the extent
to which the RNA target would be cleaved by the cleavage agents
when hybridized to the detection oligonucleotides (i.e., the probe
and invader oligonucleotides) was examined using reactions similar
to those described in Example 20, performed using
fluorescein-labeled RNA as a target.
[0610] Transcription reactions were performed as described in
Example 20 with the exception that 2% of the UTP in the reaction
was replaced with fluorescein-12-UTP (Boehringer Mannheim) and 5.3
.mu.g of the amplicon was used in a 100 .mu.l reaction. The
transcription reaction was incubated for 2.5 hours, after which the
DNA template was destroyed by the addition of 5-6 .mu.l of RQ1
RNAse-free DNAse (1 unit/.mu.l) according to the RiiboMAX.TM. kit
instructions. The organic extraction was omitted and the RNA was
collected by the addition of 10 .mu.l of 3M NaOAc, pH 5.2 and 110
.mu.l of 100% isopropanol. Following a 5 min. incubation at
4.degree. C., the precipitate was collected by centrifugation,
washed once with 80% ethanol and dried under vacuum. The resulting
RNA was dissolved in 100 .mu.l of nuclease-free water. 50% of the
sample was purified by electrophoresis through a 8%-denaturing
polyacrylamide gel (19:1 cross-linked), containing 7 M urea, in a
buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The gel slice
containing the full-length material was excised and the RNA was
eluted by soaking the slice overnight at 4.degree. C. in 200 .mu.l
of 10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA and 0.3 M NaOAc. The RNA was
then precipitated by the addition of 2.5 volumes of 100% ethanol.
After incubation at -20.degree. C. for 30 min., the precipitates
were recovered by centrifugation, washed once with 80% ethanol and
dried under vacuum. The RNA was dissolved in 25 .mu.l of
nuclease-free water and then quantitated by UV absorbance at 260
nm.
[0611] Samples of the purified RNA target were incubated for 5 or
30 min. in reactions that duplicated the Cleavase.RTM. A/G and
DNAPTth invader reactions described in Example 20 with the
exception that the reactions lacked probe and invader
oligonucleotides. Subsequent analysis of the products showed that
the RNA was very stable, with a very slight background of
non-specific degradation, appearing as a gray background in the gel
lane. The background was not dependent on the presence of enzyme in
the reaction.
[0612] Invader detection reactions using the purified RNA target
were performed using the probe/invader pair described in Example 20
(SEQ ID NOS:57 and 58). Each reaction included 500 fmole of the
target RNA, 5 pmoles of the fluorescein-labeled probe and 10 pmoles
of the invader oligonucleotide in a buffer of 10 mM MOPS, pH 7.5
with 150 mM LiCl, 4 mM MnCl.sub.2, 0.05% each Tween-20 and
Nonidet-P40 and 39 units RNAsin.RTM. (Promega). These components
were combined and warmed to 50.degree. C. and the reactions were
started by the addition of either 53 ng of Cleavase.RTM. A/G or 5
polymerase units of DNAPTth. The final reaction volume was 10
.mu.l. After 5 min at 50.degree. C., 5 .mu.l aliquots of each
reaction were removed to tubes containing 4 .mu.l of 95% formamide,
10 mM EDTA and 0.02% methyl violet. The remaining aliquot received
a drop of ChillOut.RTM. evaporation barrier and was incubated for
an additional 25 min. These reactions were then stopped by the
addition of 4 .mu.l of the above formamide solution. The products
of these reactions were resolved by electrophoresis through
separate 20% denaturing polyacrylamide gels (19:1 cross-linked),
containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4
mM EDTA. Following electrophoresis, the labeled reaction products
were visualized using the FMBIO-100 Image Analyzer (Hitachi), with
the resulting imager scans shown in FIGS. 53A (5 min reactions) and
53B (30 min. reactions).
[0613] In FIG. 53 the target RNA is seen very near the top of each
lane, while the labeled probe and its cleavage products are seen
just below the middle of each panel. The FMBIO-100 Image Analyzer
was used to quantitate the fluorescence signal in the probe bands.
In each panel, lane 1 contains products from reactions performed in
the absence of a cleavage agent, lane 2 contains products from
reactions performed using Cleavase.RTM. A/G and lane 3 contains
products from reactions performed using DNAPTth.
[0614] Quantitation of the fluorescence signal in the probe bands
revealed that after a 5 min. incubation, 12% or 300 fmole of the
probe was cleaved by the Cleavase.RTM. A/G and 29% or 700 fmole was
cleaved by the DNAPTth. After a 30 min. incubation, Cleavase.RTM.
A/G had cleaved 32% of the probe molecules and DNAPTth had cleaved
70% of the probe molecules. (The images shown in FIGS. 53A and 53B
were printed with the intensity adjusted to show the small amount
of background from the RNA degradation, so the bands containing
strong signals are saturated and therefore these images do not
accurately reflect the differences in measured fluorescence)
[0615] The data shown in FIG. 53 clearly shows that, under invasive
cleavage conditions, RNA molecules are sufficiently stable to be
detected as a target and that each RNA molecule can support many
rounds of probe cleavage.
Example 22
Titration of Target RNA in the Invader-Directed Cleavage Assay
[0616] One of the primary benefits of the invader-directed cleavage
assay as a means for detection of the presence of specific target
nucleic acids is the correlation between the amount of cleavage
product generated in a set amount of time and the quantity of the
nucleic acid of interest present in the reaction. The benefits of
quantitative detection of RNA sequences was discussed in Example
20. In this example, we demonstrate the quantitative nature of the
detection assay through the use of various amounts of target
starting material. In addition to demonstrating the correlation
between the amounts of input target and output cleavage product,
these data graphically show the degree to which the RNA target can
be recycled in this assay
[0617] The RNA target used in these reactions was the
fluorescein-labeled material described in Example 21 (i.e., SEQ ID
NO:56). Because the efficiency of incorporation of the
fluorescein-12-UTP by the T7 RNA polymerase was not known, the
concentration of the RNA was determined by measurement of
absorbance at 260 nm, not by fluorescence intensity. Each reaction
comprised 5 pmoles of the fluorescein-labeled probe (SEQ ID NO:57)
and 10 pmoles of the invader oligonudeotide (SEQ ID NO:58) in a
buffer of 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4 mM MnCl.sub.2,
0.05% each Tween-20 and Nonidet-P40 and 39 units of RNAsin.RTM.
(Promega). The amount of target RNA was varied from 1 to 100
fmoles, as indicated below. These components were combined,
overlaid with ChillOut.RTM. evaporation barrier (MJ Research) and
warmed to 50.degree. C.; the reactions were started by the addition
of either 53 ng of Cleavase.RTM. A/G or 5 polymerase units of
DNAPTth, to a final reaction volume of 10 .mu.l. After 30 minutes
at 50.degree. C., reactions were stopped by the addition of 8 .mu.l
of 95% formamide, 10 mM EDTA and 0.02% methyl violet. The unreacted
markers in lanes 1 and 2 were diluted in the same total volume (18
.mu.l). The samples were heated to 90.degree. C. for 1 minute and
2.5 .mu.l of each of these reactions were resolved by
electrophoresis through a 20% denaturing polyacrylamide gel (19:1
cross link) with 7M urea in a buffer of 45 mM Tris-Borate, pH 8.3,
1.4 mM EDTA, and the labeled reaction products were visualized
using the FMBIO-100 Image Analyzer (Hitachi), with the resulting
imager scans shown in FIG. 54.
[0618] In FIG. 54, lanes 1 and 2 show 5 pmoles of uncut probe and
500 fmoles of untreated RNA, respectively. The probe is the very
dark signal near the middle of the panel, while the RNA is the thin
line near the top of the panel. These RNAs were transcribed with a
2% substitution of fluorescein-12-UTP for natural UTP in the
transcription reaction. The resulting transcript contains 74 U
residues, which would give an average of 1.5 fluorescein labels per
molecule. With one tenth the molar amount of RNA loaded in lane 2,
the signal in lane 2 should be approximately one seventh
(0.15.times.) the fluorescence intensity of the probe in lane 1.
Measurements indicated that the intensity was closer to one
fortieth, indicating an efficiency of label incorporation of
approximately 17%. Because the RNA concentration was verified by
A260 measurement this does not alter the experimental observations
below, but it should be noted that the signal from the RNA and the
probes does not accurately reflect the relative amounts in the
reactions.
[0619] The reactions analyzed in lanes 3 through 7 contained 1, 5,
10, 50 and 100 fmoles of target, respectively, with cleavage of the
probe accomplished by Cleavase.RTM. A/G. The reactions analyzed in
lanes 8 through 12 repeated the same array of target amounts, with
cleavage of the probe accomplished by DNAPTth. The boxes seen
surrounding the product bands show the area of the scan in which
the fluorescence was measured for each reaction. The number of
fluorescence units detected within each box is indicated below each
box; background florescence was also measured.
[0620] It can be seen by comparing the detected fluorescence in
each lane that the amount of product formed in these 30 minute
reactions can be correlated to the amount of target material. The
accumulation of product under these conditions is slightly enhanced
when DNAPTth is used as the cleavage agent, but the correlation
with the amount of target present remains. This demonstrates that
the invader assay can be used as a means of measuring the amount of
target RNA within a sample.
[0621] Comparison of the fluorescence intensity of the input RNA
with that of the cleaved product shows that the invader-directed
cleavage assay creates signal in excess of the amount of target, so
that the signal visible as cleaved probe is far more intense than
that representing the target RNA. This further confirms the results
described in Example>>, in which it was demonstrated that
each RNA molecule could be used many times.
Example 23
Detection of DNA by Charge Reversal
[0622] The detection of specific targets is achieved in the
invader-directed cleavage assay by the cleavage of the probe
oligonucleotide. In addition to the methods described in the
preceding examples, the cleaved probe may be separated from the
uncleaved probe using the charge reversal technique described
below. This novel separation technique is related to the
observation that positively charged adducts can affect the
electrophoretic behavior of small oligonucleotides because the
charge of the adduct is significant relative to charge of the whole
complex. Observations of aberrant mobility due to charged adducts
have been reported in the literature, but in all cases found, the
applications pursued by other scientists have involved making
oligonucleotides larger by enzymatic extension. As the negatively
charged nucleotides are added on, the positive influence of the
adduct is reduced to insignificance. As a result, the effects of
positively charged adducts have been dismissed and have received
infinitesimal notice in the existing literature.
[0623] This observed effect is of particular utility in assays
based on the cleavage of DNA molecules. When an oligonucleotide is
shortened through the action of a Cleavase.RTM. enzyme or other
cleavage agent, the positive charge can be made to not only
significantly reduce the net negative charge, but to actually
override it, effectively "flipping" the net charge of the labeled
entity. This reversal of charge allows the products of
target-specific cleavage to be partitioned from uncleaved probe by
extremely simple means. For example, the products of cleavage can
be made to migrate towards a negative electrode placed at any point
in a reaction vessel, for focused detection without gel-based
electrophoresis. When a slab gel is used, sample wells can be
positioned in the center of the gel, so that the cleaved and
uncleaved probes can be observed to migrate in opposite directions.
Alternatively, a traditional vertical gel can be used, but with the
electrodes reversed relative to usual DNA gels (i.e., the positive
electrode at the top and the negative electrode at the bottom) so
that the cleaved molecules enter the gel, while the uncleaved
disperse into the upper reservoir of electrophoresis buffer.
[0624] An additional benefit of this type of readout is that the
absolute nature of the partition of products from substrates means
that an abundance of uncleaved probe can be supplied to drive the
hybridization step of the probe-based assay, yet the unconsumed
probe can be subtracted from the result to reduce background.
[0625] Through the use of multiple positively charged adducts,
synthetic molecules can be constructed with sufficient modification
that the normally negatively charged strand is made nearly neutral.
When so constructed, the presence or absence of a single phosphate
group can mean the difference between a net negative or a net
positive charge. This observation has particular utility when one
objective is to discriminate between enzymatically generated
fragments of DNA, which lack a 3 phosphate, and the products of
thermal degradation, which retain a 3 phosphate (and thus two
additional negative charges).
[0626] a) Characterization of the Products of Thermal Breakage Of
DNA Oligonucleotides
[0627] Thermal degradation of DNA probes results in high background
which can obscure signals generated by specific enzymatic cleavage,
decreasing the signal-to-noise ratio. To better understand the
nature of DNA thermal degradation products, we incubated the 5'
tetrachloro-fluorescein (TET)-labeled oligonucleotides 78 (SEQ ID
NO:59) and 79 (SEQ ID NO:60) (100 pmole each) in 50 .mu.l 10 mM
NaCO.sub.3 (pH 10.6), 50 mM NaCl at 90.degree. C. for 4 hours. To
prevent evaporation of the samples, the reaction mixture was
overlaid with 50 .mu.l of ChillOut.RTM. 14 liquid wax (MJ
Research). The reactions were then divided in two equal aliquots (A
and B). Aliquot A was mixed with 25 .mu.l of methyl violet loading
buffer and Aliquot B was dephosphorylated by addition of 2.5 .mu.l
of 100 mM MgCl.sub.2 and 1 .mu.l of 1 unit/.mu.l Calf Intestinal
Alkaline Phosphatase (CLAP) (Promega), with incubation at
37.degree. C. for 30 min. after which 25 .mu.l of methyl violet
loading buffer was added. One microliter of each sample was
resolved by electrophoresis through a 12% polyacrylamide denaturing
gel and imaged as described in Example 21; a 585 nm filter was used
with the FMBIO Image Analyzer. The resulting imager scan is shown
in FIG. 55. In FIG. 55, lanes 1-3 contain the TET-labeled
oligonucleotide 78 and lanes 4-6 contain the TET-labeled
oligonucleotides 79. Lanes 1 and 4 contain products of reactions
which were not heat treated. Lanes 2 and 5 contain products from
reactions which were heat treated and lanes 3 and 6 contain
products from reactions which were heat treated and subjected to
phosphatase treatment.
[0628] As shown in FIG. 55, heat treatment causes significant
breakdown of the 5'-TET-labeled DNA, generating a ladder of
degradation products (FIG. 55, lanes 2, 3, 5 and 6). Band
intensities correlate with purine and pyrimidine base positioning
in the oligonucleotide sequences, indicating that backbone
hydrolysis may occur through formation of abasic intermediate
products that have faster rates for purines then for pyrimidines
[Lindahl and Karlstrom (1973) Biochem. 12:5151].
[0629] Dephosphorylation decreases the mobility of all products
generated by the thermal degradation process, with the most
pronounced effect observed for the shorter products (FIG. 55, lanes
3 and 6). This demonstrates that thermally degraded products
possess a 3' end terminal phosphoryl group which can be removed by
dephosphorylation with CLAP. Removal of the phosphoryl group
decreases the overall negative charge by 2. Therefore, shorter
products which have a small number of negative charges are
influenced to a greater degree upon the removal of two charges.
This leads to a larger mobility shift in the shorter products than
that observed for the larger species.
[0630] The fact that the majority of thermally degraded DNA
products contain 3' end phosphate groups and Cleavase.RTM.
enzyme-generated products do not allowed the development of simple
isolation methods for products generated in the invader-directed
cleavage assay. The extra two charges found in thermal breakdown
products do not exist in the specific cleavage products. Therefore,
if one designs assays that produce specific products which contain
a net positive charge of one or two, then similar thermal breakdown
products will either be negative or neutral. The difference can be
used to isolate specific products by reverse charge methods as
shown below.
[0631] b) Dephosphorylation of Short Amino-Modified
Oligonucleotides can Reverse the Net Charge of the Labeled
Product
[0632] To demonstrate how oligonucleotides can be transformed from
net negative to net positively charged compounds, the four short
amino-modified oligonucleotides labeled 70, 74, 75 and 76 and shown
in FIGS. 56-58 were synthesized (FIG. 56 shows both
oligonucleotides 70 and 74). All four modified oligonucleotides
possess Cy-3 dyes positioned at the 5'-end which individually are
positively charged under reaction and isolation conditions
described in this example. Compounds 70 and 74 contain two amino
modified thymidines that, under reaction conditions, display
positively charged R--NH.sub.3+groups attached at the C5 position
through a C.sub.10 or C.sub.6 linker, respectively. Because
compounds 70 and 74 are 3'-end phosphorylated, they consist of four
negative charges and three positive charges. Compound 75 differs
from 74 in that the internal C.sub.6 amino modified thymidine
phosphate in 74 is replaced by a thymidine methyl phosphonate. The
phosphonate backbone is uncharged and so there are a total of three
negative charges on compound 75. This gives compound 75 a net
negative one charge. Compound 76 differs from 70 in that the
internal amino modified thymidine is replaced by an internal
cytosine phosphonate. The pK.sub.a of the N3 nitrogen of cytosine
can be from 4 to 7. Thus, the net charges of this compound, can be
from -1 to 0 depending on the pH of the solution. For the
simplicity of analysis, each group is assigned a whole number of
charges, although it is realized that, depending on the pK.sub.a of
each chemical group and ambient pH, a real charge may differ from
the whole number assigned. It is assumed that this difference is
not significant over the range of pHs used in the enzymatic
reactions studied here.
[0633] Dephosphorylation of these compounds, or the removal of the
3' end terminal phosphoryl group, results in elimination of two
negative charges and generates products that have a net positive
charge of one. In this experiment, the method of isoelectric
focusing (IEF) was used to demonstrate a change from one negative
to one positive net charge for the described substrates during
dephosphorylation.
[0634] Substrates 70, 74, 75 and 76 were synthesized by standard
phosphoramidite chemistries and deprotected for 24 hours at
22.degree. C. in 14 M aqueous ammonium hydroxide solution, after
which the solvent was removed in vacuo. The dried powders were
resuspended in 200 .mu.l of H.sub.2O and filtered through 0.2 .mu.m
filters. The concentration of the stock solutions was estimated by
UV-absorbance at 261 nm of samples diluted 200-fold in H.sub.2O
using a spectrophotometer (Spectronic Genesys 2, Milton Roy,
Rochester, N.Y.).
[0635] Dephosphorylation of compounds 70 and 74, 75 and 76 was
accomplished by treating 10 .mu.l of the crude stock solutions
(ranging in concentration from approximately 0.5 to 2 mM) with 2
units of CLAP in 100 .mu.l of CLAP buffer (Promega) at 37.degree.
C. for 1 hour. The reactions were then heated to 75.degree. C. for
15 min. in order to inactivate the CLAP. For clarity,
dephosphorylated compounds are designated `dp`. For example, after
dephosphorylation, substrate 70 becomes 70dp.
[0636] To prepare samples for IEF experiments, the concentration of
the stock solutions of substrate and dephosphorylated product were
adjusted to a uniform absorbance of 8.5.times.10.sup.-3 at 532 nm
by dilutuion with water. Two microliters of each sample were
analyzed by IEF using a PhastSystem electrophoresis unit
(Pharmacia) and PhastGel IEF 3-9 media (Pharmacia) according to the
manufacturer's protocol. Separation was performed at 15.degree. C.
with the following program: pre-run; 2,000 V, 2.5 mA, 3.5 W, 75 Vh;
load; 200 V, 2.5 mA, 3.5 W, 15 Vh; run; 2,000 V; 2.5 mA; 3.5 W, 130
Vh. After separation, samples were visualized by using the FMBIO
Image Analyzer (Hitachi) fitted with a 585 nm filter. The resulting
imager scan is shown in FIG. 59.
[0637] FIG. 59 shows results of IEF separation of substrates 70,
74, 75 and 76 and their dephosphorylated products. The arrow
labeled "Sample Loading Position" indicates a loading line, the `+`
sign shows the position of the positive electrode and the `-` sign
indicates the position of the negative electrode.
[0638] The results shown in FIG. 59 demonstrate that substrates 70,
74, 75 and 76 migrated toward the positive electrode, while the
dephosphorylated products 70dp, 74dp, 75dp and 76dp migrated toward
negative electrode. The observed differences in mobility direction
was in accord with predicted net charge of the substrates (minus
one) and the products (plus one). Small perturbations in the
mobilities of the phosphorylated compounds indicate that the
overall pIs vary. This was also true for the dephosphorylated
compounds. The presence of the cytosine in 76dp, for instance,
moved this compound further toward the negative electrode which was
indicative of a higher overall pI relative to the other
dephosphorylated compounds. It is important to note that additional
positive charges can be obtained by using a combination of natural
amino modified bases (70dp and 74dp) along with uncharged
methylphosphonate bridges (products 75dp and 76dp).
[0639] The results shown above demonstrate that the removal of a
single phosphate group can flip the net charge of an
oligonucleotide to cause reversal in an electric field, allowing
easy separation of products, and that the precise base composition
of the oligonucleotides affect absolute mobility but not the
charge-flipping effect.
Example 23
Detection of Specific Cleavage Products in the Invader-Directed
Cleavage Reaction by Charge Reversal
[0640] In this example the ability to isolate products generated in
the invader-directed cleavage assay from all other nucleic acids
present in the reaction cocktail was demonstrated using charge
reversal. This experiment utilized the following Cy3-labeled
oligonucleotide: 5'-Cy3-AminoT-AminoT-CTTTTCACCAGCGAGACGGG-3' (SEQ
ID NO:61; termed "oligo 61"). Oligo 61 was designed to release upon
cleavage a net positively charged labeled product. To test whether
or not a net positively charged 5'-end labeled product would be
recognized by the Cleavase.RTM. enzymes in the invader-directed
cleavage assay format, probe oligo 61 (SEQ ID NO:61) and invading
oligonucleotide 67 (SEQ ID NO:62) were chemically synthesized on a
DNA synthesizer (ABI 391) using standard phosphoramidite
chemistries and reagents obtained from Glen Research (Sterling,
Va.).
[0641] Each assay reaction comprised 100 fmoles of M13mp18 single
stranded DNA, 10 pmoles each of the probe (SEQ ID NO:61) and
invader (SEQ ID NO:62) oligonucleotides, and 20 units of
Cleavase.RTM. A/G in a 10 .mu.l solution of 10 mM MOPS, pH 7.4 with
100 mM KCl. Samples were overlaid with mineral oil to prevent
evaporation. The samples were brought to either 50.degree. C.,
55.degree. C., 60.degree. C., or 65.degree. C. and cleavage was
initiated by the addition of 1 .mu.l of 40 mM MnCl.sub.2. Reactions
were allowed to proceed for 25 minutes and then were terminated by
the addition of 10 .mu.l of 95% formamide containing 20 mM EDTA and
0.02% methyl violet. The negative control experiment lacked the
target M13mp18 and was run at 60.degree. C. Five microliters of
each reaction were loaded into separate wells of a 20% denaturing
polyacrylamide gel (cross-linked 29:1) with 8 M urea in a buffer
containing 45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. An electric
field of 20 watts was applied for 30 minutes, with the electrodes
oriented as indicated in FIG. 60B (i.e., in reverse orientation).
The products of these reactions were visualized using the FMBIO
fluorescence imager and the resulting imager scan is shown in FIG.
60B.
[0642] FIG. 60A provides a schematic illustration showing an
alignment of the invader (SEQ ID NO:61) and probe (SEQ ID NO:62)
along the target M13mp18 DNA; only 53 bases of the M13mp18 sequence
is shown (SEQ ID NO:63). The sequence of the inavder
oligonucleotide is displayed under the M13mp18 target and an arrow
is used above the M13mp18 sequence to indicate the position of the
invader relative to the probe and target. As shown in FIG. 60A, the
invader and probe oligonucleotides share a 2 base region of
overlap.
[0643] In FIG. 60B, lanes 1-6 contain reactions peformed at
50.degree. C., 55.degree. C., 60.degree. C., and 65.degree. C.,
respectively; lane 5 contained the control reaction (lacking
target). In FIG. 60B, the products of cleavage are seen as dark
bands in the upper half of the panel; the faint lower band seen
appears in proportion to the amount of primary product produced
and, while not limiting the invetion to a particular mechanism, may
represent cleavage one nucleotide into the duplex. The uncleaved
probe does not enter the gel and is thus not visible. The control
lane showed no detectable signal over background (lane 5). As
expected in an invasive cleavage reaction, the rate of accumulation
of specific cleavage product was temperature-dependent. Using these
particular oligonucleotides and target, the fastest rate of
accumulation of product was observed at 55.degree. C. (lane 2) and
very little product observed at 65.degree. C. (lane 4).
[0644] When incubated for extended periods at high temperature, DNA
probes can break non-specifically (i.e., suffer thermal
degradation) and the resulting fragments contribute an interfering
background to the analysis. The products of such thermal breakdown
are distributed from single-nucleotides up to the full length
probe. In this experiment, the ability of charge based separation
of cleavage products (i.e., charge reversal) would allow the
sensitve separation of the specific products of target-dependent
cleavage from probe fragments generated by thermal degradation was
examined.
[0645] To test the sensitivity limit of this detection method, the
target M13 mp18 DNA was serially diluted ten fold over than range
of 1 fmole to 1 amole. The invader and probe oligonucleotides were
those decribed above (i.e., SEQ ID NOS:61 and 62). The invasive
cleavage reactions were run as described above with the following
modifications: the reactions were performed at 55.degree. C., 250
mM or 100 mM KGlu was used in place of the 100 mM KCl and only 1
pmole of the, invader oligonucleotide was added. The reactions were
initiated as described above and allowed to progress for 12.5
hours. A negative control reaction which lacked added M13m18 target
DNA was also run. The reactions were terminated by the addition of
10 .mu.l of 95% formamide containing 20 mM EDTA and 0.02% methyl
violet, and 5 .mu.l of these mixtures were electrophoresed and
visualized as described above. The resulting imager scan is shown
in FIG. 61.
[0646] In FIG. 61, lane 1 contains the regative control; lanes 2-5
contain reactions performed using 100 mM KGlu; lanes 6-9 contain
reactions performed using 250 mM KGlu. The reactions resolved in
lanes 2 and 6 contained 1 fmole of target DNA; those in lanes 3 and
7 contained 100 amole of target; those in lanes 4 and 8 contained
10 amole of target and those in lanes 5 and 9 contained 1 amole of
target. The results shown in FIG. 61 demonstrate that the detection
limit using charge reversal to detect the production of specific
cleavage products in an invasive cleavage reaction is at or below 1
attomole or approximately 6.02.times.10.sup.5 target molecules. No
detectable signal was observed in the control lane, which indicates
that non-specific hydrolysis or other breakdown products do not
migrate in the same direction as enzyme-specific cleavage products.
The excitation and emission maxima for Cy3 are 554 and 568,
respectively, while the FMBIO Imager Analyzer excites at 532 and
detects at 585. Therefore, the limit of detection of specific
cleavage products can be improved by the use of more closely
matched excitation source and detection filters.
Example 24
Devices and Methods for the Separation and Detection of Charged
Reaction Products
[0647] This example is directed at methods and devices for
isolating and concentrating specific reaction products produced by
enzymatic reactions conducted in solution whereby the reactions
generate charged products from either a charge neutral substrate or
a substrate bearing the opposite charge borne by the specific
reaction product. The methods and devices of this example allow
isolation of, for example, the products generated by the
invader-directed cleavage assay of the present invention.
[0648] The methods and devices of this example are based on the
principle that when an electric field is applied to a solution of
charged molecules, the migration of the molecules toward the
electrode of the opposite charge occurs very rapidly. If a matrix
or other inhibitory material is introduced between the charged
molecules and the electrode of opposite charge such that this rapid
migration is dramatically slowed, the first molecules to reach the
matrix will be nearly stopped, thus allowing the lagging molecules
to catch up. In this way a dispersed population of charged
molecules in solution can be effectively concentrated into a
smaller volume. By tagging the molecules with a detectable moiety
(e.g., a fluorescent dye), detection is facilitated by both the
concentration and the localization of the analytes. This example
illustrates two embodiments of devices contemplated by the present
invention; of course, variations of these devices will be apparent
to those skilled in the art and are within the spirit and scope of
the present invention.
[0649] FIG. 62 depicts one embodiment of a device for concentrating
the positively-charged products generated using the methods of the
present invention. As shown in FIG. 62, the device comprises a
reaction tube (10) which contains the reaction solution (11). One
end of each of two thin capillaries (or other tubes with a hollow
core) (13A and 13B) are submerged in the reaction solution (11).
The capillaries (13A and 13B) may be suspended in the reaction
solution (11) such that they are not in contact with the reaction
tube itself; one appropriate method of suspending the capillaries
is to hold them in place with clamps (not shown). Alternatively,
the capillaries may be suspended in the reaction solution (11) such
that they are in contact with the reaction tube itself. Suitable
capillaries include glass capillary tubes commonly available from
scientific supply companies (e.g., Fisher Scientific or VWR
Scientific) or from medical supply houses that carry materials for
blood drawing and analysis. Though the present invention is not
limited to capillaries of any particular inner diameter, tubes with
inner diameters of up to about 1/8 inch (approximately 3 mm) are
particularly preferred for use with the present invention; for
example Kimble No. 73811-99 tubes (VWR Scientific) have an inner
diameter of 1.1 mm and are a suitable type of capillary tube.
Although the capillaries of the device are commonly composed of
glass, any nonconductive tubular material, either rigid or
flexible, that can contain either a conductive material or a
trapping material is suitable for use in the present invention. One
example of a suitable flexible tube is Tygon.RTM. clear plastic
tubing (Part No. R3603; inner diameter= 1/16 inch; outer
diameter=1/8 inch).
[0650] As illustrated in FIG. 62, capillary 13A is connected to the
positive electrode of a power supply (20) (e.g., a controllable
power supply available through the laboratory suppliers listed
above or through electronics supply houses like Radio Shack) and
capillary 13B is connected to the negative electrode of the power
supply (20). Capillary 13B is filled with a trapping material (14)
capable of trapping the positively-charged reaction products by
allowing minimal migration of products that have entered the
trapping material (14). Suitable trapping materials include, but
are not limited to, high percentage (e.g., about 20%) acrylamide
polymerized in a high salt buffer (0.5 M or higher sodium acetate
or similar salt); such a high percentage polyacrylamide matrix
dramatically slows the migration of the positively-charged reaction
products. Alternatively, the trapping material may comprise a
solid, negatively-charged matrix, such as negatively-charged latex
beads, that can bind the incoming positively-charged products. It
should be noted that any amount of trapping material (14) capable
of inhibiting any concentrating the positively-charged reaction
products may be used. Thus, while the capillary 13B in FIG. 62 only
contains trapping material in the lower, submerged portion of the
tube, the trapping material (14) can be present in the entire
capillary (13B); similarly, less trapping material (14) could be
present than that shown in FIG. 62 because the positively-charged
reaction products generally accumulate within a very small portion
of the bottom of the capillary (13B). The amount of trapping
material need only be sufficient to make contact with the reaction
solution (11) and have the capacity to collect the reaction
products. When capillary 13B is not completely filled with the
trapping material, the remaining space is filled with any
conductive material (15); suitable conductive materials are
discussed below.
[0651] By comparison, the capillary (13A) connected to the positive
electrode of the power supply 20 may be filled with any conductive
material (15; indicated by the hatched lines in FIG. 62). This may
be the sample reaction buffer (e.g., 10 mM MOPS, pH 7.5 with 150 mM
LiCl, 4 mM MnCl.sub.2), a standard electrophoresis buffer (e.g., 45
mM Tris-Borate, pH 8.3, 1.4 mM EDTA), or the reaction solution (11)
itself. The conductive material (15) is frequently a liquid, but a
semi-solid material (e.g., a gel) or other suitable material might
be easier to use and is within the scope of the present invention.
Moreover, that trapping material used in the other capillary (i.e.,
capillary 13B) may also be used as the conductive material.
Conversely, it should be noted that the same conductive material
used in the capillary (13A) attached to the positive electrode may
also be used in capillary 13B to fill the space above the region
containing the trapping material (14) (see FIG. 62).
[0652] The top end of each of the capillaries (13A and 13B) is
connected to the appropriate electrode of the power supply (20) by
electrode wire (18) or other suitable material. Fine platinum wire
(e.g., 0.1 to 0.4 mm, Aesar Johnson Matthey, Ward Hill, Mass.) is
commonly used as conductive wire because it does not corrode under
electrophoresis conditions. The electrode wire (18) can be attached
to the capillaries (13A and 13B) by a nonconductive adhesive (not
shown), such as the silicone adhesives that are commonly sold in
hardware stores for sealing plumbing fixtures. If the capillaries
are constructed of a flexible material, the electrode wire (18) can
be secured with a small hose clamp or constricting wire (not shown)
to compress the opening of the capillaries around the electrode
wire. If the conducting material (15) is a gel, an electrode wire
(18) can be embedded directly in the gel within the capillary.
[0653] The cleavage reaction is assembled in the reaction tube (10)
and allowed to proceed therein as described in proceeding examples
(e.g., Examples 22-23). Though not limited to any particular volume
of reaction solution (11), a preferred volume is less than 10 ml
and more preferably less than 0.1 ml. The volume need only be
sufficient to permit contact with both capillaries. After the
cleavage reaction is completed, an electric field is applied to the
capillaries by turning on the power source (20). As a result, the
positively-charged products generated in the course of the
invader-directed cleavage reaction which employs an
oligonucleotide, which when cleaved, generates a positively charged
fragment (described in Ex. 23) but when uncleaved bears a net
negative charge, migrate to the negative capillary, where their
migration is slowed or stopped by the trapping material (14), and
the negatively-charged uncut and thermally degraded probe molecules
migrate toward the positive electrode. Through the use of this or a
similar device, the positively-charged products of the invasive
cleavage reaction are separated from the other material (i.e.,
uncut and thermally degraded probe) and concentrated from a large
volume. Concentration of the product in a small amount of trapping
material (14) allows for simplicity of detection, with a much
higher signal-to-noise ratio than possible with detection in the
original reaction volume. Because the concentrated product is
labelled with a detectable moiety like a fluorescent dye, a
commercially-available fluorescent plate reader (not shown) can be
used to ascertain the amount of product. Suitable plate readers
include both top and bottom laser readers. Capillary 13B can be
positioned with the reaction tube (10) at any desired position so
as to accommodate use with either a top or a bottom plate reading
device.
[0654] In the alternative embodiment of the present invention
depicted in FIG. 63, the procedure described above is accomplished
by utilizing only a single capillary (13B). The capillary (13B)
contains the trapping material (14) described above and is
connected to an electrode wire (18), which in turn is attached to
the negative electrode of a power supply (20). The reaction tube
(10) has an electrode (25) embedded into its surface such that one
surface of the electrode is exposed to the interior of the reaction
tube (10) and another surface is exposed to the exterior of the
reaction tube. The surface of the electrode (25) on the exterior of
the reaction tube is in contact with a conductive surface (26)
connected to the positive electrode of the power supply (20)
through an electrode wire (18). Variations of the arrangement
depicted in FIG. 63 are also contemplated by the present invention.
For example, the electrode (25) may be in contact with the reaction
solution (11) through the use of a small hole in the reaction tube
(10); furthermore, the electrode wire (18) can be directly attached
to the electrode wire (18), thereby eliminating the conductive
surface (26).
[0655] As indicated in FIG. 63, the electrode (25) is embedded in
the bottom of a reaction tube (10) such that one or more reaction
tubes may be set on the conductive surface (26). This conductive
surface could serve as a negative electrode for multiple reaction
tubes; such a surface with appropriate contacts could be applied
through the use of metal foils (e.g., copper or platinum, Aesar
Johnson Matthey, Ward Hill, Mass.) in much the same way contacts
are applied to circuit boards. Because such a surface contact would
not be exposed to the reaction sample directly, less expensive
metals, such as the copper could be used to make the electrical
connections.
[0656] The above devices and methods are not limited to separation
and concentration of positively charged oligonucleotides. As will
be apparent to those skilled in the art, negatively charged
reaction products may be separated from neutral or positively
charged reactants using the above device and methods with the
exception that capillary 13B is attached to the positive electrode
of the power supply (20) and capillary 13A or alternatively,
electrode 25, is attached to the negative electrode of the power
supply (20).
Example 25
Primer-Directed and Primer Independent Cleavage Occur at the Same
Site when the Primer Extends to the 3' Side of a Mismatched
"Bubble" in the Downstream Duplex
[0657] As discussed above in Example 1, the presence of a primer
upstream of a bifurcated duplex can influence the site of cleavage,
and the existence of a gap between the 3' end of the primer and the
base of the duplex can cause a shift of the cleavage site up the
unpaired 5' arm of the structure (see also Lyamichev et al., supra
and U.S. Pat. No. 5,422,253). The resulting non-invasive shift of
the cleavage site in response to a primer is demonstrated in FIGS.
9, 10 and 11, in which the primer used left a 4-nucleotide gap
(relative to the base of the duplex). In FIGS. 9-11, all of the
"primer-directed" cleavage reactions yielded a 21 nucleotide
product, while the primer-independent cleavage reactions yielded a
25 nucleotide product. The site of cleavage obtained when the
primer was extended to the base of the duplex, leaving no gap was
examined. The results are shown in FIG. 64 (FIG. 64 is a
reproduction of FIG. 2C in Lyamichev et al. These data were derived
from the cleavage of the structure shown in FIG. 6, as described in
Example 1. Unless otherwise specified, the cleavage reactions
comprised 0.01 pmoles of heat-denatured, end-labeled hairpin DNA
(with the unlabeled complementary strand also present), 1 pmole
primer [complementary to the 3' arm shown in FIG. 6 and having the
sequence: 5'-GAAT TCGATTTAGGTGACACTATAGAATACA (SEQ ID NO:64)] and
0.5 units of DNAPTaq (estimated to be 0.026 pmoles) in a total
volume of 10 .mu.l of 10 mM Tris-Cl, pH 8.5, and 1.5 mM MgCl.sub.2
and 50 mM KCl. The primer was omitted from the reaction shown in
the first lane of FIG. 64 and included in lane 2. These reactions
were incubated at 55.degree. C. for 10 minutes. Reactions were
initiated at the final reaction temperature by the addition of
either the MgCl.sub.2 or enzyme. Reactions were stopped at their
incubation temperatures by the addition of 8 .mu.l of 95% formamide
with 20 mM EDTA and 0.05% marker dyes.
[0658] FIG. 64 is an autoradiogram that indicates the effects on
the site of cleavage of a bifurcated duplex structure in the
presence of a primer that extends to the base of the hairpin
duplex. The size of the released cleavage product is shown to the
left (i.e., 25 nucleotides). A dideoxynucleotide sequencing ladder
of the cleavage substrate is shown on the right as a marker (lanes
3-6).
[0659] These data show that the presence of a primer that is
adjacent to a downstream duplex (lane 2) produces cleavage at the
same site as seen in reactions performed in the absence of the
primer (lane 1) (see FIGS. 9A and B, 10B and 11A for additional
comparisons). When the 3' terminal nucleotides of the upstream
oligonucleotide can base pair to the template strand but are not
homologous to the displaced strand in the region immediately
upstream of the cleavage site (i.e., when the upstream
oligonucleotide is opening up a "bubble" in the duplex), the site
to which cleavage is apparently shifted is not wholly dependent on
the presence of an upstream oligonucleotide.
[0660] As discussed above in the Background section and in Table 1,
the requirement that two independent sequences be recognized in an
assay provides a highly desirable level of specificity. In the
invasive cleavage reactions of the present invention, the invader
and probe oligonucleotides must hybridize to the target nucleic
acid with the correct orientation and spacing to enable the
production of the correct cleavage product. When the distinctive
pattern of cleavage is not dependent on the successful alignment of
both oligonucleotides in the detection system these advantages of
independent recognition are lost.
Example 26
Invasive Cleavage and Primer-Directed Cleavage when There is Only
Partial Homology in the "X" Overlap Region
[0661] While not limiting the present invention to any particular
mechanism, invasive cleavage occurs when the site of cleavage is
shifted to a site within the duplex formed between the probe and
the target nucleic acid in a manner that is dependent on the
presence of an upstream oligonucleotide which shares a region of
overlap with the downstream probe oligonucleotide. In some
instances, the 5' region of the downstream oligonucleotide may not
be completely complementary to the target nucleic acid. In these
instances, cleavage of the probe may occur at an internal site
within the probe even in the absence of an upstream oligonucleotide
(in contrast to the base-by-base nibbling seen when a fully paired
probe is used without an invader). Invasive cleavage is
characterized by an apparent shifting of cleavage to a site within
a downstream duplex that is dependent on the presence of the
invader oligonucleotide.
[0662] A comparision between invasive cleavage and primer-directed
cleavagem may be illustrated by comparing the expected cleavage
sites of a set of probe oligonucleotides having decreasing degrees
of complementarity to the target strand in the 5' region of the
probe (i.e., the region that overlaps with the invader). A simple
test, similar to that performed on the hairpin substrate above (Ex.
25), can be performed to compare invasive cleavage with the
non-invasive primer-directed cleavage described above. Such a set
of test oligonucleotides is diagrammed in FIG. 65. The structures
shown in FIG. 65 are grouped in pairs, labeled "a", "b", "C", and
"d". Each pair has the same probe sequence annealed to the target
strand (SEQ ID NO:65), but the top structure of each pair is drawn
without an upstream oligonucleotide, while the bottom structure
includes this oligonucleotide (SEQ ID NO:66). The sequences of the
probes shown in FIGS. 64a-64d are listed in SEQ ID NOS:43, 67, 68
and 69, respectively. Probable sites of cleavage are indicated by
the black arrowheads. (It is noted that the precise site of
cleavage on each of these structures may vary depending on the
choice of cleavage agent and other experimental variables. These
particular sites are provided for illustrative purposes only.)
[0663] To conduct this test, the site of cleavage of each probe is
determined both in the presence and the absence of the upstream
oligonucleotide, in reaction conditions such as those described in
Example 19. The products of each pair of reactions are then be
compared to determine whether the fragment released from the 5' end
of the probe increases in size when the upstream oligonucleotide is
included in the reaction.
[0664] The arrangement shown in FIG. 65a, in which the probe
molecule is completely complementary to the target strand, is
similar to that shown in FIG. 32. Treatment of the top structure
with the 5' nuclease of a DNA polymerase would cause exonucleolytic
nibbling of the probe (i.e., in the absence of the upstream
oligonucleotide). In contrast, inclusion of an invader
oligonucleotide would cause a distinctive cleavage shift similar,
to those observed in FIG. 33.
[0665] The arrangements shown in FIGS. 65b and 65c have some amount
of unpaired sequence at the 5' terminus of the probe (3 and 5
bases, respectively). These small 5' arms are suitable cleavage
substrate for the 5' nucleases and would be cleaved within 2
nucleotide's of the junction between the single stranded region and
the duplex. In these arrangements, the 3' end of the upstream
oligonucleotide shares identity with a portion of the 5' region of
the probe which is complementary to the target sequence (that is
the 3' end of the invader has to compete for binding to the target
with a portion of the 5' end of the probe). Therefore, when the
upstream oligonucleotide is included it is thought to mediate a
shift in the site of cleavage into the downstream duplex (although
the present invention is not limited to any particular mechanism of
action), and this would, therefore, constitute invasive cleavage.
If the extreme 5' nucleotides of the unpaired region of the probe
were able to hybridize to the target strand, the cleavage site in
the absence of the invader might change but the addition of the
invader oligonucleotide would still shift the cleavage site to the
proper position.
[0666] Finally, in the arrangement shown in FIG. 65d, the probe and
upstream oligonucleotides share no significant regions of homology,
and the presence of the upstream oligonucleotide would not compete
for binding to the target with the probe. Cleavage of the
structures shown in FIG. 64d would occur at the same site with or
without the upstream oligonucleotide, and is thus would not
constitute invasive cleavage.
[0667] By examining any upstream oligonucleotide/probe pair in this
way, it can easily be determined whether the resulting cleavage is
invasive or merely primer-directed. Such analysis is particularly
useful when the probe is not fully complementary to the target
nucleic acid, so that the expected result may not be obvious by
simple inspection of the sequences.
[0668] From the above it is clear that the invention provides
reagents and methods to permit the detection and characterization
of nucleic acid sequences and variations in nucleic acid sequences.
The invader-directed cleavage reaction of the present invention
provides an ideal direct detection method that combines the
advantages of the direct detection assays (e.g., easy
quantification and minimal risk of carry-over contamination) with
the specificity provided by a dual oligonucleotide hybridization
assay.
[0669] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in molecular biology
or related fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
* * * * *