U.S. patent application number 09/989757 was filed with the patent office on 2002-10-24 for detection of nucleic acids by target-catalyzed product formation.
Invention is credited to Rose, Samuel J., Ullman, Edwin F., Western, Linda M..
Application Number | 20020155548 09/989757 |
Document ID | / |
Family ID | 23429108 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155548 |
Kind Code |
A1 |
Western, Linda M. ; et
al. |
October 24, 2002 |
Detection of nucleic acids by target-catalyzed product
formation
Abstract
A method is disclosed for modifying an oligonucleotide, which
method has application to the detection of a polynucleotide
analyte. An oligonucleotide is reversibly hybridized with a
polynucleotide, for example, a polynucleotide analyte, in the
presence of a 5'-nuclease under isothermal conditions. The
polynucleotide analyte serves as a recognition element to enable a
5'-nuclease to cleave the oligonucleotide to provide (i) a first
fragment-that is substantially non-hybridizable to the
polynucleotide analyte and (ii) a second fragment that lies 3' of
the first fragment (in the intact oligonucleotide) and is
substantially hybridizable to the polynucleotide analyte. At least
a 100-fold molar excess of the first fragment and/or the second
fragment are obtained relative to the molar amount of the
polynucleotide analyte. The presence of the first fragment and/or
the second fragment is detected, the presence thereof indicating
the presence of the polynucleotide analyte. The method has
particular application to the detection of a polynucleotide analyte
such as DNA. Kits for conducting methods in accordance with the
present invention are also disclosed.
Inventors: |
Western, Linda M.; (Sea
Mateo, CA) ; Rose, Samuel J.; (Los Altos, CA)
; Ullman, Edwin F.; (Atherton, CA) |
Correspondence
Address: |
Dade Behring Inc.
Legal Dept. - Patents
1717 Deerfield, Rd., #778
Deerfield
IL
60015-0778
US
|
Family ID: |
23429108 |
Appl. No.: |
09/989757 |
Filed: |
November 20, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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09989757 |
Nov 20, 2001 |
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09608721 |
Jun 30, 2000 |
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6368803 |
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09608721 |
Jun 30, 2000 |
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09440363 |
Nov 15, 1999 |
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6121001 |
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09440363 |
Nov 15, 1999 |
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09015949 |
Jan 30, 1998 |
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6110677 |
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09015949 |
Jan 30, 1998 |
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08691627 |
Aug 2, 1996 |
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5792614 |
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08691627 |
Aug 2, 1996 |
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08363169 |
Dec 23, 1994 |
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Current U.S.
Class: |
435/91.2 ;
435/6.11; 435/6.12 |
Current CPC
Class: |
C12Q 1/682 20130101;
C12Q 2521/319 20130101; C12Q 2521/301 20130101; C12Q 2521/319
20130101; C12Q 2537/149 20130101; C12Q 2563/101 20130101; C12Q
2537/149 20130101; C12Q 1/6823 20130101; C12Q 1/6823 20130101; C12Q
1/6823 20130101; C12Q 1/689 20130101; C12Q 1/6823 20130101; C12Q
1/682 20130101 |
Class at
Publication: |
435/91.2 ;
435/6 |
International
Class: |
C12P 019/34; C12Q
001/68 |
Claims
What is claimed is:
1. A method for modifying an oligonucleotide, said method
comprising incubating said oligonucleotide with a polynucleotide
and a 5'-nuclease wherein at least a portion of said
oligonucleotide is reversibly hybridized to said polynucleotide
under isothermal conditions and wherein said oligonucleotide is
cleaved to provide (i) a first fragment that is substantially
non-hybridizable to said polynucleotide and includes no more than
one nucleotide from the 5'-end of said portion and (ii) a second
fragment that is 3' of said first fragment with reference to the
intact oligonucleotide and is substantially hybridizable to said
polynucleotide.
2. The method of claim 1 wherein the amounts of fragments that are
formed are at least 100-fold larger than the amount of said
polynucleotide.
3. The method of claim 1 wherein a second oligonucleotide is
present during said incubating, said second oligonucleotide having
the characteristic of hybridizing to a site on said polynucleotide
that is 3' of the site at which said oligonucleotide is reversibly
hybridized and of being substantially non-reversibly hybridized to
said polynucleotide under said isothermal conditions.
4. The method of claim 3 wherein said second oligonucleotide
hybridizes to said polynucleotide at a site contiguous with the
site on said polynucleotide at which said first oligonucleotide
reversibly hybridizes.
5. The method of claim 4 wherein the amounts of fragments that are
formed are at least 100-fold larger than the amount of said
polynucleotide.
6. The method of claim 1 wherein a single nucleoside triphosphate
is present during said incubating.
7. A method for detecting a polynucleotide analyte, which
comprises: (a) reversibly hybridizing an oligonucleotide with a
polynucleotide analyte and a 5'-nuclease under isothermal
conditions wherein said polynucleotide analyte serves as a
recognition element to enable said 5'-nuclease to cleave said
oligonucleotide to provide (i) a first fragment that is
substantially non-hybridizable to said polynucleotide analyte and
(ii) a second fragment that lies 3' of said first fragment in the
intact oligonucleotide and is substantially hybridizable to said
polynucleotide analyte wherein at least a 100-fold molar excess of
said first fragment and/or said second fragment are obtained
relative to the molar amount of said polynucleotide analyte, and
(b) detecting the presence of said first fragment and/or said
second fragment, the presence thereof indicating the presence of
said polynucleotide analyte.
8. The method of claim 7 wherein at least one of said first
fragment and said second fragment has a label.
9. The method of claim 7 wherein said first fragment includes no
more that 1 nucleotide from the 5'-end of that portion of said
oligonucleotide that hybridizes to said polynucleotide analyte.
10. The method of claim 7 wherein a second oligonucleotide is
present during said reversible hybridizing, said second
oligonucleotide having the characteristic of hybridizing to a site
on said polynucleotide analyte that is 3' of the site at which said
oligonucleotide hybridizes wherein said polynucleotide analyte is
substantially fully hybridized to said second oligonucleotide under
said isothermal conditions.
11. The method of claim 8 wherein said oligonucleotide
hybridization sites are contiguous.
12. The method of claim 7 wherein a single nucleoside triphosphate
is present during said reversible hybridizing.
13. A method for detecting a polynucleotide analyte, said method
comprising: (a) providing in combination a medium suspected of
containing said polynucleotide analyte, a molar excess, relative to
the suspected concentration of said polynucleotide analyte, of a
first oligonucleotide at least a portion of which is capable of
reversibly hybridizing with said polynucleotide analyte under
isothermal conditions, a 5'-nuclease, and a second oligonucleotide
having the characteristic of hybridizing to a site on said
polynucleotide analyte that is 3' of the site at which said first
oligonucleotide hybridizes wherein said polynucleotide analyte is
substantially fully hybridized to said second oligonucleotide under
said isothermal conditions, (b) reversibly hybridizing under said
isothermal conditions said polynucleotide analyte and said first
oligonucleotide, wherein said first oligonucleotide is cleaved as a
function of the presence of said polynucleotide analyte to provide,
in at least a 100-fold molar excess of said polynucleotide analyte,
(i) a first fragment that is substantially non-hybridizable to said
polynucleotide analyte and/or (ii) a second fragment that is 3' of
said first fragment in said first oligonucleotide and is
substantially hybridizable to said polynucleotide analyte, and (c)
detecting the presence of said first fragment and/or said second
fragment, the presence thereof indicating the presence of said
polynucleotide analyte.
14. The method of claim 13 wherein said first fragment and/or said
second fragment has a label.
15. The method of claim 14 wherein said label is selected from the
group consisting of a member of a specific binding pair, dyes,
fluorescent molecules, chemiluminescers, coenzymes, enzyme
substrates, radioactive groups and suspendible particles.
16. The method of claim 13 wherein said polynucleotide analyte is
DNA.
17. The method of claim 13 wherein said first fragment includes no
more than 1 nucleotide from the 5'-end of that portion of said
first oligonucleotide that is capable of hybridizing to said
polynucleotide analyte.
18. The method of claim 13 wherein said second oligonucleotide
hybridizes to said polynucleotide at a site contiguous with the
site on said polynucleotide at which said first oligonucleotide
hybridizes.
19. The method of claim 13 wherein a single nucleoside triphosphate
is present in said combination during said reversible
hybridizing.
20. A method for detecting a DNA analyte, said method comprising:
(a) providing in combination a medium suspected of containing said
DNA analyte, a first oligonucleotide at least a portion of which is
capable of reversibly hybridizing with said DNA analyte under
isothermal conditions, a 5'-nuclease, and a second oligonucleotide
having the characteristic of hybridizing to a site on said DNA
analyte that is 3' of the site at which said first oligonucleotide
hybridizes wherein said DNA analyte is substantially fully
hybridized to said second oligonucleotide under said isothermal
conditions, (b) reversibly hybridizing said polynucleotide analyte
and said first oligonucleotide under said isothermal conditions,
wherein said first oligonucleotide is cleaved to (i) a first
fragment that is substantially non-hybridizable to said DNA analyte
and (ii) a second fragment that is 3' of said first fragment in
said first oligonucleotide and is substantially hybridizable to
said DNA analyte wherein at least a 100-fold molar excess, relative
to said DNA analyte, of said first fragment and/or said second
fragment is produced and (c) detecting the presence of said first
fragment and/or said second fragment, the presence thereof
indicating the presence of said DNA analyte.
21. The method of claim 20 wherein said first oligonucleotide has a
substituent that facilitates separation of said first fragment or
said second fragment from said medium.
22. The method of claim 20 wherein first fragment and/or said
second fragment has a label.
23. The method of claim 22 wherein said label is selected from the
group consisting of a member of a specific binding pair, dyes,
fluorescent molecules, chemiluminescers, coenzymes, enzyme
substrates, radioactive groups and suspendible particles.
24. The method of claim 20 wherein a single nucleoside triphosphate
is present in said combination during said reversible
hybridizing.
25. The method of claim 20 wherein said second oligonucleotide
hybridizes to said polynucleotide at a site contiguous with the
site on said polynucleotide at which said first oligonucleotide
hybridizes.
26. The method of claim 20 wherein said first oligonucleotide
and/or said second oligonucleotide is DNA.
27. A method for detecting a polynucleotide analyte, said method
comprising: (a) providing in combination a medium suspected of
containing said polynucleotide analyte, a first DNA oligonucleotide
at least a portion of which is capable of reversibly hybridizing
with said polynucleotide analyte under isothermal conditions, a
5'-nuclease, and a second DNA oligonucleotide having the
characteristic of hybridizing to a site on said polynucleotide
analyte that is 3' of, and contiguous with, the site at which said
first DNA oligonucleotide hybridizes wherein said polynucleotide
analyte is substantially fully hybridized to said second DNA
oligonucleotide under said isothermal conditions, (b) reversibly
hybridizing under said isothermal conditions said polynucleotide
analyte and said first DNA oligonucleotide, wherein said first DNA
oligonucleotide is cleaved as a function of the presence of said
polynucleotide analyte to provide, in at least a 100-fold molar
excess of said polynucleotide analyte, (i) a first fragment that is
substantially non-hybridizable to said polynucleotide analyte
and/or (ii) a second fragment that is 3' of said first fragment in
said first DNA oligonucleotide and is substantially hybridizable to
said polynucleotide analyte, and (c) detecting the presence of said
first fragment and/or said second fragment, the presence thereof
indicating the presence of said polynucleotide analyte.
28. The method of claim 27 wherein said first fragment and/or said
second fragment has a label.
29. The method of claim 28 wherein said label is selected from the
group consisting of a member of a specific binding pair, dyes,
fluorescent molecules, chemiluminescers, coenzymes, enzyme
substrates, radioactive groups and suspendible particles.
30. The method of claim 27 wherein said polynucleotide analyte is
DNA.
31. The method of claim 27 wherein a single nucleoside triphosphate
is present in said combination during said reversible
hybridizing.
32. A kit for detection of a polynucleotide comprising in packaged
combination: (a) a first oligonucleotide having the characteristic
that, when reversibly hybridized under isothermal conditions to at
least a portion of said polynucleotide, it is degraded by a
5'-nuclease to provide (i) a first fragment that is substantially
non-hybridizable to said polynucleotide and (ii) a second fragment
that is 3' of said first fragment in said first oligonucleotide and
is substantially hybridizable to said polynucleotide (b) a second
oligonucleotide having the characteristic of hybridizing to a site
on said polynucleotide that is separated by no more than one
nucleotide from the 3'-end of the site at which said first
oligonucleotide hybridizes wherein said polynucleotide is
substantially fully hybridized to said second oligonucleotide under
said isothermal conditions, and (c) a 5'-nuclease.
33. The kit of claim 32 which comprises a single nucleoside
triphosphate.
34. The kit of claim 32 wherein said first oligonucleotide and said
second oligonucleotide are DNA.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention.
[0002] Nucleic acid hybridization has been employed for
investigating the identity and establishing the presence of nucleic
acids. Hybridization is based on complementary base pairing. When
complementary single stranded nucleic acids are incubated together,
the complementary base sequences pair to form double stranded
hybrid molecules. The ability of single stranded deoxyribonucleic
acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded
structure with a complementary nucleic acid sequence has been
employed as an analytical tool in molecular biology research. The
availability of radioactive nucleoside triphosphates of high
specific activity and the .sup.32P labelling of DNA with T4
polynucleotide kinase has made it possible to identify, isolate,
and characterize various nucleic acid sequences of biological
interest. Nucleic acid hybridization has great potential in
diagnosing disease states associated with unique nucleic acid
sequences. These unique nucleic acid sequences may result from
genetic or environmental change in DNA by insertions, deletions,
point mutations, or by acquiring foreign DNA or RNA by means of
infection by bacteria, molds, fungi, and viruses. Nucleic acid
hybridization has, until now, been employed primarily in academic
and industrial molecular biology laboratories. The application of
nucleic acid hybridization as a diagnostic tool in clinical
medicine is limited because of the frequently very low
concentrations of disease related DNA or RNA present in a patient's
body fluid and the unavailability of a sufficiently sensitive
method of nucleic acid hybridization analysis.
[0003] Current methods for detecting specific nucleic acid
sequences generally involve immobilization of the target nucleic
acid on a solid support such as nitrocellulose paper, cellulose
paper, diazotized paper, or a nylon membrane. After the target
nucleic acid is fixed on the support, the support is contacted with
a suitably labelled probe nucleic acid for about two to forty-eight
hours. After the above time period, the solid support is washed
several times at a controlled temperature to remove unhybridized
probe. The support is then dried and the hybridized material is
detected by autoradiography or by spectrometric methods.
[0004] When very low concentrations must be detected, the current
methods are slow and labor intensive, and nonisotopic labels that
are less readily detected than radiolabels are frequently not
suitable. A method for increasing the sensitivity to permit the use
of simple, rapid, nonisotopic, homogeneous or heterogeneous methods
for detecting nucleic acid sequences is therefore desirable.
[0005] Recently, a method for the enzymatic amplification of
specific segments of DNA known as the polymerase chain reaction
(PCR) method has been described. This in vitro amplification
procedure uses two or more different oligonucleotide primers for
different strands of the target nucleic acid and is based on
repeated cycles of denaturation, oligonucleotide primer annealing,
and primer extension by thermophilic polymerase, resulting in the
exponential increase in copies of the region flanked by the
primers. The different PCR primers, which anneal to opposite
strands of the DNA, are positioned so that the polymerase catalyzed
extension product of one primer can serve as a template strand for
the other primer, leading to the accumulation of discrete fragments
whose length is defined by the distance between the 5'-ends of the
oligonucleotide primers.
[0006] Other methods for amplifying nucleic acids are single primer
amplification, ligase chain reaction (LCR), nucleic acid sequence
based amplification (NASBA) and the Q-beta-replicase method.
Regardless of the amplification used, the amplified product must be
detected.
[0007] Depending on which of the above amplification methods are
employed, the methods generally employ from seven to twelve or more
reagents. Furthermore, the above methods provide for exponential
amplification of a target or a reporter oligonucleotide.
Accordingly, it is necessary to rigorously avoid contamination of
assay solutions by the amplified products to avoid false positives.
Some of the above methods require expensive thermal cycling
instrumentation and additional reagents and sample handling steps
are needed for detection of the amplified product.
[0008] Most assay methods that do not incorporate amplification of
a target DNA avoid the problem of contamination, but they are not
adequately sensitive or simple. Some of the methods involve some
type of size discrimination such as electrophoresis, which adds to
the complexity of the methods.
[0009] One method for detecting nucleic acids is to employ nucleic
acid probes. One method utilizing such probes is described in U.S.
Pat. No. 4,868,104, the disclosure of which is incorporated herein
by reference. A nucleic acid probe may be, or may be capable of
being, labeled with a reporter group or may be, or may be capable
of becoming, bound to a support.
[0010] Detection of signal depends upon the nature of the label or
reporter group. If the label or reporter group is an enzyme,
additional members of the signal producing system include enzyme
substrates and so forth. The product of the enzyme reaction is
preferably a luminescent product, or a fluorescent or
non-fluorescent dye, any of which can be detected
spectrophotometrically, or a product that can be detected by other
spectrometric or electrometric means. If the label is a fluorescent
molecule, the medium can be irradiated and the fluorescence
determined. Where the label is a radioactive group, the medium can
be counted to determine the radioactive count.
[0011] It is desirable to have a sensitive, simple method for
detecting nucleic acids. The method should minimize the number and
complexity of steps and reagents. The need for sterilization and
other steps needed to prevent contamination of assay mixtures
should be avoided.
[0012] 2. Description of the Related Art.
[0013] Methods for detecting nucleic acid sequences are discussed
by Duck, et al., in U.S. Pat. No. 5,011,769 and corresponding
International Patent Application WO 89/10415. A method of cleaving
a nucleic acid molecule is disclosed in European Patent Application
0 601 834 A1 (Dahlberg, et al.).
[0014] Holland, et al., Clinical Chemistry (1992) 38:462-463,
describe detection of specific polymerase chain reaction product by
utilizing the 5' to 3' exonuclease activity of Thermus aquaticus
DNA polymerase. Longley, et al., Nucleic Acids Research (1990)
18:7317-7322, discuss characterization of the 5' to 3' exonuclease
associated with Thermus aquaticus DNA polymerase. Lyamichev, et
al., Science (1993) 260:778-783, disclose structure-specific
endonucleolytic cleavage of nucleic acids by eubacterial DNA
polymerases.
[0015] A process for amplifying, detecting and/or cloning nucleic
acid sequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202,
4,800,159, 4,965,188 and 5,008,182. Sequence polymerization by
polymerase chain reaction is described by Saiki, et al., (1986)
Science, 230: 1350-1354. Primer-directed enzymatic amplification of
DNA with a thermostable DNA polymerase is described by Saiki, et
al., Science (1988) 239:487.
[0016] U.S. patent applications Ser. Nos. 07/299,282 and
07/399,795, filed Jan. 19, 1989, and Aug. 29, 1989, respectively,
describe nucleic acid amplification using a single polynucleotide
primer. The disclosures of these applications are incorporated
herein by reference including the references listed in the sections
entitled "Description of the Related Art."
[0017] Other methods of achieving the result of a nucleic acid
amplification are described by Van Brunt in Bio/Technology (1990)
8(No.4): 291-294. These methods include ligase chain reaction
(LCR), nucleic acid sequence based amplification (NASBA) and
Q-beta-replicase amplification of RNA. LCR is also discussed in
European Patent Applications Nos. 439,182 (Backman I) and 473,15
(Backman II).
[0018] NASBA is a promoter-directed, isothermal enzymatic process
that induces in vitro continuous, homogeneous and isothermal
amplification of specific nucleic acid.
[0019] Q-beta-replicase relies on the ability of Q-beta-replicase
to amplify its RNA substrate exponentially under isothermal
conditions.
[0020] Another method for conducting an amplification of nucleic
acids is referred to as strand displacement amplification (SDA).
SDA is an isothermal, in vitro DNA amplification technique based on
the ability of a restriction enzyme to nick the unmodified strand
of a hemiphosphorothioate form of its restriction site and the
ability of a DNA polymerase to initiate replication at the nick and
displace the downstream nontemplate strand intact. Primers
containing the recognition sites for the nicking restriction enzyme
drive the exponential amplification.
[0021] Another amplification procedure for amplifying nucleic acids
is known as 3SR, which is an RNA specific target method whereby RNA
is amplified in an isothermal process combining promoter directed
RENA polymerase, reverse transcriptase and RNase H with target
RNA.
SUMMARY OF THE INVENTION
[0022] One aspect of the present invention is a method for
modifying an oligonucleotide. The method comprises incubating the
oligonucleotide with a polynucleotide and a 5'-nuclease wherein at
least a portion of the oligonucleotide is reversibly hybridized to
the polynucleotide under isothermal conditions. The oligonucleotide
is cleaved to provide (i) a first fragment that is substantially
non-hybridizable to the polynucleotide and includes no more than
one nucleotide from the 5'-end of the portion and (ii) a second
fragment that is 3' of the first fragment with reference to the
intact oligonucleotide and is substantially hybridizable to the
polynucleotide.
[0023] Another aspect of the present invention is a method for
detecting a polynucleotide analyte. An oligonucleotide is
reversibly hybridized with a polynucleotide analyte and a
5'-nuclease under isothermal conditions. The polynucleotide analyte
serves as a recognition element to enable a 5'-nuclease to cleave
the oligonucleotide to provide (i) a first fragment that is
substantially non-hybridizable to the polynucleotide analyte and
(ii) a second fragment that lies 3' of the first fragment (in the
intact oligonucleotide) and is substantially hybridizable to the
polynucleotide analyte. At least a 100-fold molar excess of the
first fragment and/or the second fragment are obtained relative to
the molar amount of the polynucleotide analyte. The presence of the
first fragment and/or the second fragment is detected, the presence
thereof indicating the presence of the polynucleotide analyte.
[0024] Another embodiment of the present invention is a method for
detecting a polynucleotide analyte. A combination is provided
comprising a medium suspected of containing the polynucleotide
analyte, an excess, relative to the suspected concentration of the
polynucleotide analyte, of a first oligonucleotide at least a
portion of which is capable of reversibly hybridizing with the
polynucleotide analyte under isothermal conditions, a 5'-nuclease,
and a second oligonucleotide having the characteristic of
hybridizing to a site on the polynucleotide analyte that is 3' of
the site at which the first oligonucleotide hybridizes. The
polynucleotide analyte is substantially fully hybridized to the
second oligonucleotide under such isothermal conditions. The
polynucleotide is reversibly hybridized under the isothermal
conditions to the first oligonucleotide, which is cleaved as a
function of the presence of the polynucleotide analyte to provide,
in at least a 100-fold molar excess of the polynucleotide analyte,
(i) a first fragment that is substantially non-hybridizable to the
polynucleotide analyte and/or (ii) a second fragment that lies 3'
of the first fragment (in the intact first oligonucleotide) and is
substantially hybridizable to the polynucleotide analyte. The
presence of the first fragment and/or the second fragment is
detected, the presence thereof indicating the presence of the
polynucleotide analyte.
[0025] Another embodiment of the present invention is a method for
detecting a DNA analyte. A combination is provided comprising a
medium suspected of containing the DNA analyte, a first
oligonucleotide at least a portion of which is capable of
reversibly hybridizing with the DNA analyte under isothermal
conditions, a 5'-nuclease, and a second oligonucleotide having the
characteristic of hybridizing to a site on the DNA analyte that is
3' of the site at which the first oligonucleotide hybridizes. The
DNA analyte is substantially fully hybridized to the second
oligonucleotide under isothermal conditions. The polynucleotide
analyte is reversibly hybridized to the first oligonucleotide under
isothermal conditions. The first oligonucleotide is cleaved to (i)
a first fragment that is substantially non-hybridizable to the DNA
analyte and (ii) a second fragment that lies 3' of the first
fragment (in the intact first oligonucleotide) and is substantially
hybridizable to the DNA analyte. At least a 100-fold molar excess,
relative to the DNA analyte, of the first fragment and/or the
second fragment is produced. The presence of the first fragment
and/or the second fragment is detected, the presence thereof
indicating the presence of the DNA analyte.
[0026] Another embodiment of the present invention is a kit for
detection of a polynucleotide. The kit comprises in packaged
combination (a) a first oligonucleotide having the characteristic
that, when reversibly hybridized under isothermal conditions to the
polynucleotide, it is degraded by a 5'-nuclease to provide (i) a
first fragment that is substantially non-hybridizable to the
polynucleotide and (ii) a second fragment that is 3' of the first
fragment (in the first oligonucleotide) and is substantially
hybridizable to the polynucleotide, (b) a second oligonucleotide
having the characteristic of hybridizing to a site on the
polynucleotide that is separated by no more than one nucleotide
from the 3'-end of the site at which the first oligonucleotide
hybridizes wherein the polynucleotide is substantially fully
hybridized to the second oligonucleotide under the isothermal
conditions, and (c) a 5'-nuclease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1-3 are schematics of different embodiments in
accordance with the present invention.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0028] The present invention permits catalyzed cleavage of an
oligonucleotide that is modulated by a portion of a polynucleotide
analyte, such as a polynucleotide, that is comprised of a target
polynucleotide sequence to which a portion of the oligonucleotide
hybridizes. As such, the methods of the present invention provide
for very high sensitivity assays for polynucleotide analytes. The
methods are simple to conduct and no temperature cycling is
required. Consequently, no expensive thermal cycling
instrumentation is needed. Furthermore, only a few reagents are
used, thus further minimizing cost and complexity of an assay. In
addition, the absence of amplified products, which are potential
amplification targets, permits the use of less rigorous means to
avoid contamination of assay solutions by target sequences that
could produce false positives.
[0029] Before proceeding further with a description of the specific
embodiments of the present invention, a number of Ha terms will be
defined.
[0030] Polynucleotide analyte--a compound or composition to be
measured that is a polymeric nucleotide, which in the intact
natural state can have about 20 to 500,000 or more nucleotides and
in an isolated state can have about 30 to 50,000 or more
nucleotides, usually about 100 to 20,000 nucleotides, more
frequently 500 to 10,000 nucleotides. Isolation of analytes from
the natural state, particularly those having a large number of
nucleotides, frequently results in fragmentation. The
polynucleotide analytes include nucleic acids from any source in
purified or unpurified form including DNA (dsDNA and ssDNA) and
RNA, including t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA,
chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof,
genes, chromosomes, plasmids, the genomes of biological material
such as microorganisms, e.g., bacteria, yeasts, viruses, viroids,
molds, fungi, plants, animals, humans, and fragments thereof, and
the like. Preferred polynucleotide analytes are double stranded DNA
(dsDNA) and single stranded DNA (ssDNA). The polynucleotide analyte
can be only a minor fraction of a complex mixture such as a
biological sample. The analyte can be obtained from various
biological material by procedures well known in the art. Some
examples of such biological material by way of illustration and not
limitation are disclosed in Table I below.
1TABLE I Microorganisms of interest include: Corynebacteria
Corynebacterium diphtheria Pneumococci Diplococcus pneumoniae
Streptococci Streptococcus pyrogenes Streptococcus salivarus
Staphylococci Staphylococcus aureus Staphylococcus albus Neisseria
Neisseria meningitidis Neisseria gonorrhea Enterobacteriaciae
Escherichia coil Aerobacter aerogenes The colliform Klebsiella
pneumoniae bacteria Salmonella typhosa Salmonella choleraesuis The
Salmonellae Salmonella typhimurium Shigella dysenteria The
Shigellae Shigella schmitzii Shigella arabinotarda Shigella
flexneri Shigella boydii Shigella sonnei Other enteric bacilli
Proteus vulgaris Proteus species Proteus mirabilis Proteus morgani
Pseudowonas aeruginosa Alcaligenes faecalis Vibrio cholerae
Hemophilus-Bordetella group Rhizopus oryzae Hemophilus influenza,
H. ducryi Rhizopus arrhizua Phycomycetes Hemophilus hemophilus
Rhizopus nigricans Hemophilus aegypticus Sporotrichum schenkii
Hernophilus parainfluenza Flonsecaea pedrosoi Bordetella pertussis
Fonsecacea compact Pasteurellae Fonsecacea dermatidis Pasteurella
pestis Cladosporium carrionii Pasteurella tulareusis Phialophora
verrucosa Brucellae Aspergillus nidulans Brucella melitensis
Madurella mycetomi Brucella abortus Madurella grisea Brucella suis
Allescheria boydii Aerobic Spore-forming Bacilli Phialophora
jeanselmei Bacillus anthracis Microsporum gypseum Bacillus subtills
Trichophyton mentagrophytes Bacillus megateriwn Keratinomyces
ajelloi Bacillus cereus Microsporum canis Anaerobic Spore-forming
Bacilli Trichophyton rubrum Clostridium botulinum Microsporum
adouini Clostridium tetani Viruses Clostridium perfringens
Adenoviruses Clostridium novyi Herpes Viruses Clostridium septicum
Herpes simplex Clostridium histolyticum Varicella (Chicken pox)
Clostridium tertium Herpes Zoster (Shingles) Clostridium
bifermentans Virus B Clostridium sporogenes Cytomegalovirus
Mycobacteria Pox Viruses Mycobacterium tuberculosis Variola
(smallpox) hominis Vaccinia Mycobacterium bovis Poxvirus bovis
Mycobacterium avium Paravaccinia Mycobacteruim leprae Molluscum
contagiosum Mycobacterium paratuberculosis Picornaviruses
Actinomycetes (fungus-like bacteria) Poliovirus Actinomyces Isaeli
Coxsackievirus Actinomyces bovis Echoviruses Actinomyces naeslundii
Rhinoviruses Nocardia asteroides Myxoviruses Nocardia brasiliensis
Influenza(A, B, The Spirochetes and C) Treponema pallidum
Parainfluenza (1-4) Streptobacillus monoiliformis Mumps Virus
Treponema pertenue Newcastle Disease Virus Spirillumn minus Measles
Virus Treponema carateum Rinderpest Virus Borrelia recurrentis
Canine Distemper Virus Leptospira icterohemorrhagiae Respiratory
Syncytial Virus Leptospira canicola Rubella Virus Tryanasomes
Arboviruses Mycoplasmas Eastern Equine Encephalitis Virus
Mycoplasma pneumoniae Western Equine Encephalitis Virus Other
pathogens Sindbis Virus Listeria monocytogenes Chikugunya Virus
Erysipelothrix rhusiopathiae Semliki Forest Virus Streptobacillus
moniliformis Mayora Virus Donvania granulomatis St. Louis
Encephalitis Virus Bartonella bacilliformis California Encephalitis
Virus Rickettsiae (bacteria-like Colorado Tick Fever Virus
parasites) Yellow Fever Virus Rickettsia prowazekii Dengue Virus
Rickettsia mooseri Reoviruses Rickettsia rickettsii Reovirus Types
1-3 Rickettsia conori Retroviruses Rickettsia australis Human
Immunodeficiency Viruses Rickettsia sibiricus (HIV) Rickettsia
akari Human T-cell Lymphotrophic Rickettsia tsutsugamushi Virus I
& II (HTLV) Rickettsia burnetti Hepatitis Rickettsia quintana
Hepatitis A Virus Chlamydia (unclassifiable parasites Hepatitis B
Virus bacterial/viral) Hepatitis nonA-nonB Virus Chlamydia agents
(naming uncertain) Tumor Viruses Fungi Rauscher Leukemia Virus
Cxyptococcus neoformans Gross Virus Blastomyces dermatidis Maloney
Leukemia Virus Hisoplasma capsulatum Human Papilloma Virus
Coccidioides immitis Paracoccidicides brasiliensis Candida albicans
Aspergilius fumigatus Mucor coryrabifer (Absidia corymbifera)
[0031] The polynucleotide analyte, where appropriate, may be
treated to cleave the analyte to obtain a polynucleotide that
contains a target polynucleotide sequence, for example, by shearing
or by treatment with a restriction endonuclease or other site
specific chemical cleavage method. However, it is an advantage of
the present invention that the polynucleotide analyte can be used
in its isolated state without further cleavage.
[0032] For purposes of this invention, the polynucleotide analyte,
or a cleaved polynucleotide obtained from the polynucleotide
analyte, will usually be at least partially denatured or single
stranded or treated to render it denatured or single stranded. Such
treatments are well-known in the art and include, for instance,
heat or alkali treatment. For example, double stranded DNA can be
heated at 90-100.degree. C. for a period of about 1 to 10 minutes
to produce denatured material.
[0033] 3'- or 5'-End of an oligonucleotide--as used herein this
phrase refers to a portion of an oligonucleotide comprising the 3'-
or 5'-terminus, respectively, of the oligonucleotide.
[0034] 3'- or 5'-Terminus of an oligonucleotide--as used herein
this term refers to the terminal nucleotide at the 3'- or 5'-end,
respectively, of an oligonucleotide.
[0035] Target polynucleotide sequence--a sequence of nucleotides to
be identified, which may be the polynucleotide analyte but is
usually existing within a polynucleotide comprising the
polynucleotide analyte. The identity of the target polynucleotide
sequence is known to an extent sufficient to allow preparation of
an oligonucleotide having a portion or sequence that hybridizes
with the target polynucleotide sequence. In general, when one
oligonucleotide is used, the oligonucleotide hybridizes with the
5'-end of the target polynucleotide sequence. When a second
oligonucleotide is used, it hybridizes to a site on the target
polynucleotide sequence that is 3' of the site to which the first
oligonucleotide hybridizes. (It should be noted that the
relationship can be considered with respect to the double stranded
molecule formed when the first and second oligonucleotides are
hybridized to the polynucleotide. In such context the second
oligonucleotide is 5-primeward of the first oligonucleotide with
respect to the "strand" comprising the first and second
oligonucleotides.) The relationships described above are more
clearly seen with reference to FIG. 3. The target polynucleotide
sequence usually contains from about 10 to 1,000 nucleotides,
preferably 15 to 100 nucleotides, more preferably, 20 to 70
nucleotides. The target polynucleotide sequence is part of a
polynucleotide that may be the entire polynucleotide analyte. The
minimum number of nucleotides in the target polynucleotide sequence
is selected to assure that the presence of target polynucleotide
sequence in a sample is a specific indicator of the presence of
polynucleotide analyte in a sample. Very roughly, the sequence
length is usually greater than about 1.6 log L nucleotides where L
is the number of base pairs in the genome of the biologic source of
the sample. The number of nucleotides in the target sequence is
usually the sum of the lengths of those portions of the
oligonucleotides that hybridize with the target sequence plus the
number of nucleotides lying between the portions of the target
sequence that hybridize with the oligonucleotides.
[0036] Oligonucleotide--a polynucleotide, usually a synthetic
polynucleotide, usually single stranded that is constructed such
that at least a portion thereof hybridizes with the target
polynucleotide sequence of the polynucleotide. The oligonucleotides
of this invention are usually 10 to 150 nucleotides, preferably,
deoxyoligonucleotides of 15 to 100 nucleotides, more preferably, 20
to 60 nucleotides, in length.
[0037] The first oligonucleotide, or "the" oligonucleotide when a
second oligonucleotide is not employed, has a 5'-end about 0 to 100
nucleotides, preferably, 1 to 20 nucleotides in length that does
not hybridize with the target polynucleotide sequence and usually
has a 10 to 40 nucleotide sequence that hybridizes with the target
polynucleotide sequence. In general, the degree or amplification is
reduced somewhat as the length of the portion of the
oligonucleotide that does not hybridize with the target
polynucleotide sequence increases. The first oligonucleotide also
may have a sequence at its 3'-end that does not hybridize with the
target polynucleotide sequence.
[0038] The second oligonucleotide preferably hybridizes at its
3'-end with the target polynucleotide sequence at a site on the
target polynucleotide sequence 3' of the site of binding of the
first oligonucleotide. The length of the portion of the second
oligonucleotide that hybridizes with the target polynucleotide
sequence is usually longer than the length of the portion of the
first oligonucleotide that hybridizes with the target
polynucleotide sequence and is usually 20 to 100 nucleotides. The
melting temperature of the second oligonucleotide hybridized to the
target polynucleotide sequence is preferably at least as high, more
preferably, at least 5.degree. C. higher than the melting
temperature of the first oligonucleotide hybridized to the target
polynucleotide sequence.
[0039] The oligonucleotides can be oligonucleotide mimics such a
polynucleopeptides, phosphorothioates or phosphonates except that
the first oligonucleotide usually has at least one phosphodiester
bond to the nucleoside at the 5'-end of the sequence that
hybridizes with the target polynucleotide sequence. When
oligonucleotide mimics are used that provide very strong binding,
such as polynucleopeptides, the length of the portion of the second
oligonucleotide that hybridizes with the target polynucleotide
sequence may be reduced to less than 20 and, preferably, greater
than 10.
[0040] Various techniques can be employed for preparing an
oligonucleotide or other polynucleotide utilized in the present
invention. They can be obtained by biological synthesis or by
chemical synthesis. For short oligonucleotides (up to about 100
nucleotides) chemical synthesis will frequently be more economical
as compared to biological synthesis. In addition to economy,
chemical synthesis provides a convenient way of incorporating low
molecular weight compounds and/or modified bases during the
synthesis step. Furthermore, chemical synthesis is very flexible in
the choice of length and region of the target polynucleotide
sequence. The oligonucleotides can be synthesized by standard
methods such as those used in commercial automated nucleic acid
synthesizers. Chemical synthesis of DNA on a suitably modified
glass or resin results in DNA covalently attached to the surface.
This may offer advantages in washing and sample handling. For
longer sequences standard replication methods employed in molecular
biology can be used such as the use of M13 for single stranded DNA
as described by J. Messing (1983) Methods Enzymol, 101, 20-78.
[0041] In addition to standard cloning techniques, in vitro
enzymatic methods may be used such as polymerase catalyzed
reactions. For preparation of RNA, T7 RNA polymerase and a suitable
DNA template can be used. For DNA, polymerase chain reaction (PCR)
and single primer amplification are convenient.
[0042] Other chemical methods of polynucleotide or oligonucleotide
synthesis include phosphotriester and phosphodiester methods
(Narang, et al., Meth. Enzymol (1979) 68: 90) and synthesis on a
support (Beaucage, et al., Tetrahedron (1981) Letters 22:
1859-1862) as well as phosphoramidate techniques, Caruthers, M. H.,
et al., "Methods in Enzymology," Vol. 154, pp. 287-314 (1988), and
others described in "Synthesis and Applications of DNA and RNA," S.
A. Narang, editor, Academic Press, New York, 1987, and the
references contained therein.
[0043] Fragment--in general, in the present method the
oligonucleotide (or the first oligonucleotide when a second
oligonucleotide is employed) is cleaved only when at least a
portion thereof is reversibly hybridized with a target
polynucleotide sequence and, thus, the target polynucleotide
sequence acts as a recognition element for cleavage of the
oligonucleotide, thereby yielding two portions. One fragment is
substantially non-hybridizable to the target polynucleotide
sequence. The other fragment is substantially hybridizable to the
target polynucleotide sequence and 3' of the other fragment with
respect to the oligonucleotide in its uncleaved form.
[0044] 5'-Nuclease--a sequence-independent deoxyribonuclease enzyme
that catalyzes the cleavage of an oligonucleotide into fragments
only when at least a portion of the oligonucleotide is hybridized
to the target polynucleotide sequence. The enzyme selectively
cleaves the oligonucleotide near the 5'-terminus of the bound
portion, within 5 nucleotides thereof, preferably within 1 to 2
nucleotides thereof and does not cleave the unhybridized
oligonucleotide or the target polynucleotide sequence. Such enzymes
include both 5'-exonucleases and 5'-endonucleases but exclude
ribonucleases such as RNAse H and restriction enzymes. 5'-nucleases
useful in the present invention must be stable under the isothermal
conditions used in the present method and are usually thermally
stable nucleotide polymerases having 5'-exonuclease activity such
as Taq DNA polymerase (e.g. AmpliTaq(TM) from Perkin-Elmer
Corporation, Norwalk, N.J.), Thermalase Tbr(TM) DNA polymerase
(from Amresco, Solon, Ohio), Ultra Therm(TM) DNA polymerase (from
Bio/Can Scientific, Ontario, Canada), Replitherm (TM) DNA
polymerase (from Epicentre, Madison, Wis.), Tfl (TM) DNA polymerase
(from Epicentre), Panozyme(TM) DNA polymerase (from Panorama
Research, Mountain View, Calif.), Tth(TM) DNA polymerase (from
Epicentre), rBst (TM) DNA polymerase (from Epicentre), Heat
Tuff(TM) DNA polymerase (from Clontech, Palo Alto, Calif.), and the
like, derived from any source such as cells, bacteria, such as E.
coli, plants, animals, virus, thermophilic bacteria, and so forth
wherein the polymerase may be modified chemically or through
genetic engineering to provide for thermal stability and/or
increased activity.
[0045] Isothermal conditions--a uniform or constant temperature at
which the modification of the oligonucleotide in accordance with
the present invention is carried out. The temperature is chosen so
that the duplex formed by hybridizing the oligonucleotide to a
polynucleotide with a target polynucleotide sequence is in
equilibrium with the free or unhybridized oligonucleotide and free
or unhybridized target polynucleotide sequence, a condition that is
otherwise referred to herein as "reversibly hybridizing" the
oligonucleotide with a polynucleotide. Normally, at least 1%,
preferably 20 to 80%, usually less than 95% of the polynucleotide
is hybridized to the oligonucleotide under the isotermal
conditions. Accordingly, under isothermal conditions there are
molecules of polynucleotide that are hybridized with the
oligonucleotide, or portions thereof, and are in dynamic
equilibrium with molecules that are not hybridized with the
oligonucleotide. Some fluctuation of the temperature may occur and
still achieve the benefits of the present invention. The
fluctuation generally is not necessary for carrying out the methods
of the present invention and usually offer no substantial
improvement. Accordingly, the term "isothermal conditions" includes
the use of a fluctuating temperature, particularly random or
uncontrolled fluctuations in temperature, but specifically excludes
the type of fluctuation in temperature referred to as thermal
cycling, which is employed in some known amplification procedures,
e.g., polymerase chain reaction.
[0046] Polynucleotide primer(s) or oligonucleotide primer(s)--an
oligonucleotide that is usually employed in a chain extension on a
polynucleotide template.
[0047] Nucleoside triphosphates--nucleosides having a
5'-triphosphate substituent. The nucleosides are pentose sugar
derivatives of nitrogenous bases of either purine or pyrimidine
derivation, covalently bonded to the 1'-carbon of the pentose
sugar, which is usually a deoxyribose or a ribose. The purine bases
include adenine(A), guanine(G), inosine, and derivatives and
analogs thereof. The pyrimidine bases include cytosine (C), thymine
(T), uracil (U), and derivatives and analogs thereof. Nucleoside
triphosphates include deoxyribonucleoside triphosphates such as
DATP, dCTP, dGTP and dTTP and ribonucleoside triphosphates such as
rATP, rCTP, rGTP and rUTP. The term "nucleoside triphosphates" also
includes derivatives and analogs thereof.
[0048] Nucleotide--a base-sugar-phosphate combination that is the
monomeric unit of nucleic acid polymers, i.e., DNA and RNA.
[0049] Nucleoside--is a base-sugar combination or a nucleotide
lacking a phosphate moiety.
[0050] Nucleotide polymerase--a catalyst, usually an enzyme, for
forming an extension of an oligonucleotide along a polynucleotide
template where the extension is complementary thereto. The
nucleotide polymerase is a template dependent polynucleotide
polymerase and utilizes nucleoside triphosphates as building blocks
for extending the 3'-end of a oligonucleotide to provide a sequence
complementary with the single stranded portion of the
polynucleotide to which the oligonucleotide is hybridized to form a
duplex.
[0051] Hybridization (hybridizing) and binding--in the context of
nucleotide sequences these terms are used interchangeably herein.
The ability of two nucleotide sequences to hybridize with each
other is based on the degree of complementarity of the two
nucleotide sequences, which in turn is based on the fraction of
matched complementary nucleotide pairs. The more nucleotides in a
given sequence that are complementary to another sequence, the more
stringent the conditions can be for hybridization and the more
specific will be the binding of the two sequences. Increased
stringency is achieved by elevating the temperature, increasing the
ratio of cosolvents, lowering the salt concentration, and the
like.
[0052] Homologous or substantially identical--In general, two
polynucleotide sequences that are identical or can each hybridize
to the same polynucleotide sequence are homologous. The two
sequences are homologous or substantially identical where the
sequences each have at least 90%, preferably 100%, of the same or
analogous base sequence where thymine (T) and uracil (U) are
considered the same. Thus, the ribonucleotides A, U, C and G are
taken as analogous to the deoxynucleotides dA, dT, dC, and dG,
respectively. Homologous sequences can both be DNA or one can be
DNA and the other RNA.
[0053] Complementary--Two sequences are complementary when the
sequence of one can bind to the sequence of the other in an
anti-parallel sense wherein the 3'-end of each sequence binds to
the 5'-end of the other sequence and each A, T(U), G, and C of one
sequence is then aligned with a T(U), A, C, and G, respectively, of
the other sequence.
[0054] Copy--means a sequence that is a direct identical or
homologous copy of a single stranded polynucleotide sequence as
differentiated from a sequence that is complementary to the
sequence of such single stranded polynucleotide.
[0055] Member of a specific binding pair ("sbp member")--one of two
different molecules, having an area on the surface or in a cavity
which specifically binds to, and is thereby defined as
complementary with, a particular spatial and polar organization of
the other molecule. The members of the specific binding pair are
referred to as ligand and receptor (antiligand). These may be
members of an immunological pair such as antigen-antibody, or may
be operator-repressor, nuclease-nucleotide, biotin-avidin,
hormones-hormone receptors, nucleic acid duplexes, IgG-protein A,
DNA-DNA, DNA-RNA, and the like.
[0056] Ligand--any compound for which a receptor naturally exists
or can be prepared.
[0057] Receptor ("antiligand")--any compound or composition capable
of recognizing a particular spatial and polar organization of a
molecule, e.g., epitopic or determinant site. Illustrative
receptors include naturally occurring receptors, e.g., thyroxine
binding globulin, antibodies, enzymes, Fab fragments, lectins,
nucleic acids, repressors, protection enzymes, protein A,
complement component Clq, DNA binding proteins or ligands and the
like.
[0058] Small organic molecule--a compound of molecular weight less
than 1500, preferably 100 to 1000, more preferably 300 to 600 such
as biotin, fluorescein, rhodamine and other dyes, tetracycline and
other protein binding molecules, and haptens, etc. The small
organic molecule can provide a means for attachment of a nucleotide
sequence to a label or to a support or may itself be a label.
[0059] Support or surface--a porous or non-porous water insoluble
material. The support can be hydrophilic or capable of being
rendered hydrophilic and includes inorganic powders such as silica,
magnesium sulfate, and alumina; natural polymeric materials,
particularly cellulosic materials and materials derived from
cellulose, such as fiber containing papers, e.g., filter paper,
chromatographic paper, etc.; synthetic or modified naturally
occurring polymers, such as nitrocellulose, cellulose acetate, poly
(vinyl chloride), polyacrylamide, cross linked dextran, agarose,
polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),
polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,
poly(vinyl butyrate), etc.; either used by themselves or in
conjunction with other materials; glass available as Bioglass,
ceramics, metals, and the like. Natural or synthetic assemblies
such as liposomes, phospholipid vesicles, and cells can also be
employed.
[0060] Binding of sbp members to a support or surface may be
accomplished by well-known techniques, commonly available in the
literature. See, for example, "Immobilized Enzymes," Ichiro
Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol.
Chem., 245:3059 (1970). The surface can have any one of a number of
shapes, such as strip, rod, particle, including bead, and the
like.
[0061] Label or reporter group or reporter molecule--a member of a
signal producing system. Usually the label or reporter group or
reporter molecule is conjugated to or becomes bound to, or
fragmented from, an oligonucleotide or to a nucleoside triphosphate
and is capable of being detected directly or, through a specific
binding reaction, and can produce a detectible signal. In general,
any label that is detectable can be used. The label can be isotopic
or nonisotopic, usually non-isotopic, and can be a catalyst, such
as an enzyme or a catalytic polynucleotide, promoter, dye,
fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate,
radioactive group, a small organic molecule, amplifiable
polynucleotide sequence, a particle such as latex or carbon
particle, metal sol, crystallite, liposome, cell, etc., which may
or may not be further labeled with a dye, catalyst or other
detectible group, and the like. Labels include an oligonucleotide
or specific polynucleotide sequence that can provide a template for
amplification or ligation or act as a ligand such as for a
repressor protein. The label is a member of a signal producing
system and can generate a detectable signal either alone or
together with other members of the signal producing system. The
label can be bound directly to a nucleotide sequence or can become
bound thereto by being bound to an sbp member complementary to an
sbp member that is bound to a nucleotide sequence.
[0062] Signal Producing System--The signal producing system may
have one or more components, at least one component being the label
or reporter group or reporter molecule. The signal producing system
generates a signal that relates to the presence or amount of target
polynucleotide sequence or a polynucleotide analyte in a sample.
The signal producing system includes all of the reagents required
to produce a measurable signal. When the label is not conjugated to
a nucleotide sequence, the label is normally bound to an sbp member
complementary to an sbp member that is bound to, or part of, a
nucleotide sequence. Other components of the signal producing
system may be included in a developer solution and can include
substrates, enhancers, activators, chemiluminescent compounds,
cofactors, inhibitors, scavengers, metal ions, specific binding
substances required for binding of signal generating substances,
and the like. Other components of the signal producing system may
be coenzymes, substances that react with enzymic products, other
enzymes and catalysts, and the like. The signal producing system
provides a signal detectable by external means, by use of
electromagnetic radiation, desirably by visual examination. The
signal-producing system is described more fully in U.S. patent
application Ser. No. 07/555,323, filed Jul. 19, 1990, the relevant
disclosure of which is incorporated herein by reference.
[0063] Amplification of nucleic acids or polynucleotides--any
method that results in the formation of one or more copies of a
nucleic acid or a polynucleotide molecule, usually a nucleic acid
or polynucleotide analyte, or complements thereof, present in a
medium.
[0064] Exponential amplification of nucleic acids or
polynucleotides--any method that results in the formation of one or
more copies of a nucleic acid or polynucleotide molecule, usually a
nucleic acid or polynucleotide analyte, present in a medium.
[0065] Methods for the enzymatic amplification of specific double
stranded sequences of DNA include those described above such as the
polymerase chain reaction (PCR), amplification of a single stranded
polynucleotide using a single polynucleotide primer, ligase chain
reaction (LCR), nucleic acid sequence based amplification (NASBA),
Q-beta-replicase method, strand displacement amplification (SDA),
and 3SR.
[0066] Conditions for carrying out an amplification, thus, vary
depending upon which method is selected. Some of the methods such
as PCR utilize temperature cycling to achieve denaturation of
duplexes, oligonucleotide primer annealing, and primer extension by
thermophilic template dependent polynucleotide polymerase. Other
methods such as NASBA, Q-beta-replicase method, SDA and 3SR are
isothermal. As can be seen, there are a variety of known
amplification methods and a variety of conditions under which these
methods are conducted to achieve exponential amplification.
[0067] Linear amplification of nucleic acids or
polynucleotides--any method that results in the formation of one or
more copies of only the complement of a nucleic acid or
polynucleotide molecule, usually a nucleic acid or polynucleotide
analyte, present in a medium. Thus, one difference between linear
amplification and exponential amplification is that the latter
produces copies of the polynucleotide whereas the former produces
only the complementary strand of the polynucleotide. In linear
amplification the number of complements formed is, in principle,
directly proportional to-the time of the reaction as opposed to
exponential amplification wherein the number of copies is, in
principle, an exponential function of the time or the number of
temperature cycles.
[0068] Ancillary Materials--various ancillary materials will
frequently be employed in the methods and assays carried out in
accordance with the present invention. For example, buffers will
normally be present in the assay medium, as well as stabilizers for
the assay medium and the assay components. Frequently, in addition
to these additives, proteins may be included, such as albumins,
organic solvents such as formamide, quaternary ammonium salts,
polycations such as dextran sulfate, surfactants, particularly
non-ionic surfactants, binding enhancers, e.g., polyalkylene
glycols, or the like.
[0069] As mentioned above, the present invention has a primary
application to methods for detecting a polynucleotide analyte. In
one aspect of the invention an oligonucleotide is reversibly
hybridized with a polynucleotide analyte in the presence of a
5'-nuclease under isothermal conditions. In this way the
polynucleotide analyte serves as a "recognition element" to enable
the 5'-nuclease to specifically cleave the oligonucleotide to
provide first and second fragments when the oligonucleotide is
reversibly hybridized to the polynucleotide analyte. The first
fragment comprises the 5'-end of the oligonucleotide (with
reference to the intact or original oligonucleotide) and is
substantially non-hybridizable to the polynucleotide analyte and
can serve as a label. The first fragment generally includes at
least a portion of that part the 5'-end of the original
oligonucleotide that was not hybridized to the polynucleotide
analyte when the portion of the oligonucleotide that is
hybridizable with the polynucleotide analyte is reversibly
hybridized thereto. Additionally, the first fragment may include
nucleotides (usually, no more than 5, preferably, no more than 2,
more preferably, no more than 1 of such nucleotides) that are
cleaved by the 5'-nuclease from the 5'-end of that portion (or
sequence) of the original oligonucleotide that was hybridized to
the polynucleotide analyte. Therefore, it is in the above context
that the first fragment is "substantially non-hybridizable" with
the polynucleotide analyte. The second fragment comprises the
sequence of nucleotides at the 3'-end of the oligonucleotide that
were reversibly hybridized to the polynucleotide analyte minus
those nucleotides cleaved by the 5'-nuclease when the original
oligonucleotide is reversibly hybridized to the polynucleotide
analyte. Accordingly, the second fragment is "substantially
hybridizable" to the polynucleotide analyte having resulted from
that portion of the oligonucleotide that reversibly hybridizes with
the polynucleotide analyte.
[0070] As mentioned above, the 3'-end of the oligonucleotide may
include one or more nucleotides that do not hybridize with the
polynucleotide analyte and may comprise a label. At least a
100-fold molar excess of the first fragment and/or the second
fragment are obtained relative to the molar amount of the
polynucleotide analyte. The sequence of at least one of the
fragments is substantially Preserved during the reaction. The
presence of the first fragment and/or the second fragment is
detected, the presence thereof indicating the presence of the
polynucleotide analyte.
[0071] The 5'-nuclease is generally present in an amount sufficient
to cause the cleavage of the oligonucleotide, when it is reversibly
hybridized to the polynucleotide analyte, to proceed at least half
as rapidly as the maximum rate achievable with excess enzyme,
preferably, at least 75% of the maximum rate. The concentration of
the 5'-nuclease is usually determined empirically. Preferably, a
concentration is used that is sufficient such that further increase
in the concentration does not decrease the time for the
amplification by over 5-fold, preferably 2-fold. The primary
limiting factor generally is the cost of the reagent. In this
respect, then, the polynucleotide analyte, or at least the target
polynucleotide sequence, and the enzyme are generally present in a
catalytic amount.
[0072] The oligonucleotide that is cleaved by the enzyme is usually
in large excess, preferably, 10.sup.-9 M to 10.sup.-5 M, and is
used in an amount that maximizes the overall rate of its cleavage
in accordance with the present invention wherein the rate is at
least 10%, preferably, 50%, more preferably, 90%, of the maximum
rate of reaction possible. Concentrations of the oligonucleotide
lower than 50% may be employed to facilitate detection of the
fragment(s) produced in accordance with the present invention. The
amount of oligonucleotide is at least as great as the number of
molecules of product desired. Usually, the, concentration of the
oligonucleotide is 0.1 nanomolar to 1 millimolar, preferably, 1
nanomolar to 10 micromolar. It should be noted that increasing the
concentration of the oligonucleotide causes the reaction rate to
approach a limiting value that depends on the oligonucleotide
sequence, the temperature, the concentration of the target
polynucleotide sequence and the enzyme concentration. For many
detection methods very high concentrations of the oligonucleotide
may make detection more difficult.
[0073] The amount of the target polynucleotide sequence that is to
be copied can be as low as one or two molecules in a sample but
generally may vary from about 10.sup.2 to 10.sup.10, more usually
from about 10.sup.3 to 10.sup.8 molecules in a sample preferably at
least 10.sup.-21M in the sample and may be 10.sup.-10 to
10.sup.-19M, more usually 10.sup.-14 to 10.sup.-19M.
[0074] In carrying out the methods in accordance with the present
invention, an aqueous medium is employed. Other polar solvents may
also be employed as cosolvents, usually oxygenated organic solvents
of from 1-6, more usually from 1-4, carbon atoms, including
alcohols, ethers and the like. Usually these cosolvents, if used,
are present in less than about 70 weight percent, more usually in
less than about 30 weight percent.
[0075] The pH for the medium is usually in the range of about 4.5
to 9.5, more usually in the range of about 5.5-8.5, and preferably
in the range of about 6-8. The pH and temperature are chosen so as
to achieve the reversible hybridization or equilibrium state under
which cleavage of an oligonucleotide occurs in accordance with the
present invention. In some instances, a compromise is made in the
reaction parameters in order to optimize the speed, efficiency, and
specificity of these steps of the present method. Various buffers
may be used to achieve the desired pH and maintain the pH during
the determination. Illustrative buffers include borate, phosphate,
carbonate, Tris, barbital and the like. The particular buffer
employed is not critical to this invention but in individual
methods one buffer may be preferred over another.
[0076] As mentioned above the reaction in accordance with the
present invention is carried out under isothermal conditions. The
reaction is generally carried out at a temperature that is near the
melting temperature of the oligonucleotide:polynucleotide analyte
complex. Accordingly, the temperature employed depends on a number
of factors. Usually, for cleavage of the oligonucleotide in
accordance with the present invention, the temperature is about
35.degree. C. to 90.degree. C. depending on the length and sequence
of the oligonucleotide. It will usually be desired to use
relatively high temperature of 60.degree. C. to 85.degree. C. to
provide for a high rate of reaction. The amount of the fragments
formed depends on the incubation time and-temperature. In general,
a moderate temperature is normally employed for carrying out the
methods. The exact temperature utilized also varies depending on
the salt concentration, pH, solvents used, and the length of and
composition of the target polynucleotide sequence as well as the
oligonucleotide as mentioned above.
[0077] One embodiment of the invention is depicted in FIG. 1.
Oligonucleotide OL is combined with polynucleotide analyte PA
having target polynucleotide sequence TPS and with a 5'-nuclease,
which can be, for example, a Taq polymerase. In this embodiment OL
is labeled (*) within what is designated the first fragment,
produced upon cleavage of the oligonucleotide in accordance with
the present invention. OL in this embodiment usually is at least 10
nucleotides in length, preferably, about 10 to 50 nucleotides in
length, more preferably, 15 to 30 or more nucleotides in length. In
general, the length of OL should be sufficient so that a portion
hybridizes with TPS, the length of such portion approximating the
length of TPS. In this embodiment the length of OL is chosen so
that the cleavage of no more than 5, preferably, no more than 1 to
3, more preferably, 1 to 2 nucleotides, therefrom results in two
fragments. The first fragment, designated LN, is no more than 5
nucleotides in length, preferably, 1 to 3 nucleotides in length,
more preferably, 1 to 2 nucleotides in length and the second
fragment, designated DOL, is no more than 5, preferably, no more
than 1 to 3, more preferably, no more than 1 to 2, nucleotides
shorter than the length of OL.
[0078] As shown in FIG. 1, OL hybridizes with TPS to give duplex I.
The hybridization is carried out under isothermal conditions so
that OL is reversibly hybridized with TPS. OL in duplex I is
cleaved to give DOL and LN, wherein LN includes a labeled
nucleotide (*). In the embodiment depicted in FIG. 1, DOL is the
complement of TPS except for the nucleotides missing at the 5'-end.
Since during the course of the isothermal reaction the 5'-end of PA
may be cleaved at or near the 5'-end of TPS, DOL may also have 0 to
5 nucleotides at its 3'-end that overhang and cannot hybridize with
the residual portion of TPS. The isothermal conditions are chosen
such that equilibrium exists between duplex I and its single
stranded components, namely, PA and OL. Upon cleavage of OL within
duplex I, an equilibrium is also established between duplex I and
its single stranded components, PA and DOL. Since OL is normally
present in large excess relative to the amount of DOL formed in the
reaction, there are usually many more duplexes containing OL than
DOL. The reaction described above for duplex I continuously
produces additional molecules of DOL.
[0079] The reaction is allowed to continue until a sufficient
number of molecules of DOL and LN are formed to permit detection of
the labeled LN (LN*) and, thus, the polynucleotide analyte. In this
way the enzyme-catalyzed cleavage of nucleotides from the 5'-end of
OL is modulated by and, therefore, related to the presence of the
polynucleotide analyte. Depending on the amount of PA present, a
sufficient number of molecules for detection can be obtained where
the time of reaction is from about 1 minute to 24 hours.
Preferably, the reaction can be carried out in less than 5 hours.
As a matter of convenience it is usually desirable to minimize the
time period as long as the requisite of number of molecules of
detectable fragment is achieved. In general, the time period for a
given degree of cleavage can be minimized by optimizing the
temperature of the reaction and using concentrations of the
5'-nuclease and the oligonucleotide that provide reaction rates
near the maximum achievable with excess of these reagents.
Detection of the polynucleotide analyte is accomplished indirectly
by detecting the label in fragment LN*. Alternatively, DOL may be
detected, for example, by using the label as a means of separating
LN* and OL from the reaction mixture and then detecting the
residual DOL.
[0080] Detection of the labeled fragment is facilitated in a number
of ways. For example, a specific pair member such as biotin or a
directly detectable label such a fluorescein can be used. The low
molecular weight LN* can be separated by electrophoresis, gel
exclusion chromatography, thin layer chromatography ultrafiltration
and the like and detected by any convenient means such as a
competitive binding assay or direct detection of the label.
Alternatively, the oligonucleotide can be labeled within the second
(DOL) fragment with a specific binding member such as a ligand, a
small organic molecule, a polynucleotide sequence or a protein, or
with a directly detectable label such as a directly detectable
small organic molecules, e.g., fluorescein, a sensitizer, a
coenzyme and the like. Detection will then depend on
differentiating the oligonucleotide with labels on both ends from
singly labeled fragments where one labeled end has been cleaved. In
this case it is desirable to label one end of OL with a specific
binding member that facilitates removal of OL and the fragment
retaining the label by using a complementary sbp member bound to a
support. The residual labeled fragments bearing the other label are
then detected by using a method appropriate for detecting that
label.
[0081] One method for detecting nucleic acids is to employ nucleic
acid probes. Other assay formats and detection formats are
disclosed in U.S. patent applications Ser. Nos. 07/229,282 and
07/399,795 filed Jan. 19, 1989, and Aug. 29, 1989, respectively,
U.S. patent application Ser. No. 07/555,323 filed Jul. 19, 1990,
U.S. patent application Ser. No. 07/555,968 and U.S. patent
application Ser. No. 07/776,538 filed Oct. 11, 1991, which have
been incorporated herein by reference.
[0082] Examples of particular labels or reporter molecules and
their detection can be found in U.S. patent application Ser. No.
07/555,323 filed Jul. 19, 1990, the relevant disclosure of which is
incorporated herein by reference.
[0083] Detection of the signal will depend upon the nature of the
signal producing system utilized. If the label or reporter group is
an enzyme, additional members of the signal producing system
include enzyme substrates and so forth. The product of the enzyme
reaction is preferably a luminescent product, or a fluorescent or
non-fluorescent dye, any of which can be detected
spectrophotometrically, or a product that can be detected by other
spectrometric or electrometric means. If the label is a fluorescent
molecule, the medium can be irradiated and the fluorescence
determined. Where the label is a radioactive group, the medium can
be counted to determine the radioactive count.
[0084] Another embodiment of the present invention is depicted in
FIG. 2. Oligonucleotide OL' has a first portion or sequence SOL1
that is not hybridized to TPS' and a second portion or sequence
SOL2 that is hybridized to TPS'. OL' is combined with
polynucleotide analyte PA' having target polynucleotide sequence
TPS' and with a 5'-endonuclease (5'-endo), which can be, for
example, Taq DNA polymerase and the like. OL' and 5'-endo are
generally present in concentrations as described above. In the
embodiment of FIG. 2, OL' is labeled (*) within the sequence SOL1
wherein SOL1 may intrinsically comprise the label or may be
extrinsically labeled with a specific binding member or directly
detectable labeled. The length of SOL2 is as described in the
embodiment of FIG. 1. In general, the length of SOL2 should be
sufficient to hybridize with TPS', usually approximating the length
of TPS'. SOL1 may be any length as long as it does not
substantially interfere with the cleavage of OL' and will
preferably be relatively short to avoid such interference. Usually,
SOL1 is about 1 to 100 nucleotides in length, preferably, 8 to 20
nucleotides in length.
[0085] In this embodiment the cleavage of SOL1 from SOL2 results in
two fragments. Cleavage in SOL2 occurs within 5 nucleotides of the
bond joining SOL1 and SOL2 in OL'. The exact location of cleavage
is not critical so long as the enzyme cleaves OL' only when it is
bound to TPS'. The two fragments are designated LNSOL1 and DSOL2.
LNSOL1 is comprised of the 5'-end of OL' and DSOL2 is comprised of
the 3'-end of OL'. The sequence of at least one of LNSOL1 and DSOL2
remains substantially intact during the cleavage reaction. As shown
in FIG. 2, SOL2 of OL' hybridizes with TPS' to give duplex I'. The
hybridization is carried out under isothermal conditions so that
OL' is reversibly hybridized with TPS'. OL' in duplex I' is cleaved
to give DSOL2 and LNSOL1, the latter of which comprises a label. In
the embodiment depicted in FIG. 2, DSOL2 is the complement of TPS'
except for any nucleotides missing at the 5'-end thereof as a
result of the cleavage of the cleavage reaction and any nucleotides
appended to the 3'-end of OL' (not shown in FIG. 2) that do not
hybridize with TPS'.
[0086] The isothermal conditions are chosen such that equilibrium
exists between duplex I' and its single stranded components, i.e.,
PA' and OL'. Upon cleavage of OL' within duplex I' and equilibrium
is also established between duplex I' and its single stranded
components, PA' and DSOL2. Since OL' is normally present in large
excess relative to the amount of DSOL2 formed in the reaction,
there are usually many more duplexes containing OL' than DSOL2. The
reaction described above for duplex I' continuously produces
molecules of DSOL2 and LNSOL1. The reaction is allowed to continue
until a sufficient number of molecules of DSOL2 and LNSOL1 are
formed to permit detection of one or both of these fragments. In
this way the enzyme-catalyzed cleavage of LNSOL1 from the 5'-end of
the portion of OL' hybridized to PA' is modulated by, and therefore
related to, the presence of the polynucleotide analyte. The
reaction parameters and the detection of DSOL2 and/or LNSOL1 are
generally as described above for the embodiment of FIG. 1.
[0087] Various ways of controlling the cleavage of the
oligonucleotide can be employed. For example, the point of cleavage
can be controlled by introducing a small organic group, such as
biotin, into the nucleotide at the 5'-terminus of OL' or the
nucleotide in SOL2 that is at the junction of SOL2 and SOL1.
[0088] An embodiment using a second oligonucleotide is depicted in
FIG. 3. The second oligonucleotide (OL2) hybridizes to a site TPS2
on PA" that lies 3' of the site of hybridization (TPS1) of the
sequence SOL2" of the first oligonucleotide, namely, OL". In the
embodiment shown OL2 fully hybridizes with TPS2. This is by way of
example and not limitation. The second oligonucleotide can include
nucleotides at its 5' end that are not hybridizable with the target
polynucleotide sequence, but its 3'-end is preferably hybridizable.
Preferably, OL2 binds to a site (TPS2) that is contiguous with the
site to which SOL2" hybridizes (TPS1). However, it is within-the
purview of the present invention that the second oligonucleotide
hybridize with PA" within 1 to 5 nucleotides, preferably, 1
nucleotide, of the site to which SOL2" hybridizes. The second
oligonucleotide, OL2, is usually at least as long as, and
preferably longer than, SOL2", preferably, at least 2 nucleotides
longer than SOL2". In general, the second oligonucleotide is about
20-100 nucleotides in length, preferably, 30-80 nucleotides in
length depending on the length of SOL2". Normally, the second
oligonucleotide is chosen such that it dissociates from duplex I"
at a higher temperature than that at which OL" dissociates, usually
at least 3.degree. C., preferably, at least 5.degree. C. or more
higher.
[0089] The presence of OL2 in duplex I" can effect the site of
cleavage of OL". In particular, when OL2 binds to PA" that is not
contiguous with the SOL2" site of hybridization, the cleavage site
may be shifted one or more nucleotides.
[0090] The concentration of the second oligonucleotide employed in
this embodiment is usually at least 1 picomolar, but is preferably
above 0.1 nanomolar to facilitate rapid binding to PA", more
preferably, at least 1 nanomolar to 1 micromolar. In accordance
with the embodiment of FIG. 3, OL" in duplex I" is cleaved by
5'-endo to give DSOL2" and LNSOL1". The reaction is permitted to
continue until the desired number of molecules of labeled fragment
are formed. The reaction parameters and detection of DSOL2" and/or
LNSOL1" are similar to those described above for the embodiment of
FIG. 1.
[0091] In general and specifically in any of the embodiments of
FIGS. 1 to 3 above, the 3'-end of the first oligonucleotide, for
example, OL, OL' and OL", may have one or more nucleotides that do
not hybridize with the target polynucleotide sequence and can serve
as a label but need not do so.
[0092] It is also within the purview of the present invention to
employ a single nucleoside triphosphate in any of the above
embodiments, depending on the particular 5'-endonuclease chosen for
the above cleavage. The decision to use a nucleoside triphosphate
and the choice of the nucleoside triphosphate are made empirically
based on its ability to accelerate the reaction in accordance with
the present invention. The nucleoside triphosphate is preferably
one that cannot be incorporated into the first oligonucleotide as a
consequence of the binding of the oligonucleotide to the target
polynucleotide sequence. In this particular embodiment the added
nucleoside triphosphate is present in a concentration of 1
micromolar to 10 millimolar, preferably, 10 micromolar to 1
millimolar, more preferably, 100 micromolar to 1 millimolar. It is
also within the purview of the present invention to utilize the
added nucleoside triphosphate to chain extend the 3'-terminus of
the second oligonucleotide to render it contiguous with the site on
the target polynucleotide sequence at which the first
oligonucleotide hybridizes. In this approach the second
oligonucleotide serves as a polynucleotide primer for chain
extension. In addition, the nucleoside triphosphate is
appropriately selected to accomplish such chain extension and the
5'-nuclease is selected to also have template-dependent nucleotide
polymerase activity. In any event such an approach is primarily
applicable to the situation where the site of binding of this
second oligonucleotide, TPS2, is separated from the site of binding
of the first oligonucleotide, TPS1, by a sequence of one or more
identical bases that are complementary to the added nucleotide
triphosphate.
[0093] In the embodiment of FIG. 3 the mixture containing PA", OL",
the second oligonucleotide OL2 and the nucleoside triphosphate is
incubated at an appropriate isothermal temperature at which OL" and
PA" are in equilibrium with duplex I" wherein most of the molecules
of PA" and duplex I" are hybridized to OL2. During the time when a
molecule of OL" is bound to PA", the 5'-endo causes the cleavage by
hydrolysis of OL' in accordance with the present invention. When
the remaining portion of cleaved oligonucleotide (DSOL2")
dissociates from PA", an intact molecule of OL" becomes hybridized,
whereupon the process is repeated.
[0094] In one experiment in accordance with the above embodiment,
incubation for 3 hours at 72.degree. C. resulted in the production
of over 10.sup.12 molecules of DSOL2" and LNSOL1", which was over
10.sup.4 increase over the number of molecules of PA" that was
present initially in the reaction mixture. OL" was labeled with a
.sup.32P-phosphate at the 5'-terminus. The cleaved product LNSOL1"
was detected by applying the mixture to an electrophoresis gel and
detecting a band that migrated more rapidly than the band
associated with OL". The appearance of this band was shown to be
associated with the presence and amount of PA" where a minimum of
10.sub.8 molecules of PA" was detected.
[0095] Alternative approaches for detection of LNSOL1"and/or DSOL2"
may also be employed in the above embodiment. For example, in one
approach biotin is attached to any part of SOL2" that is cleaved
from OL" by the 5'-endonuclease. The fragment DSOL2" and OL"
containing the biotin are separated from LNSOL1", for example, by
precipitation with streptavidin and filtration. The unprecipitated
labeled fragment LNSOL1" is then detected by any standard binding
assay, either without separation (homogeneous) or with separation
(heterogeneous) of any of the assay components or products.
[0096] Homogeneous immunoassays are exemplified by enzyme
multiplied immunoassay techniques ("EMIT") disclosed in Rubenstein,
et al., U.S. Pat. No. 3,817,837, column 3, line 6 to column 6, line
64; immunofluorescence methods such as those disclosed in Ullman,
et al., U.S. Pat. No. 3,996,345, column 17, line 59 to column 23,
line 25; enzyme channeling techniques such as those disclosed in
Maggio, et al., U.S. Pat. No. 4,233,402, column 6, line 25 to
column 9, line 63; and other enzyme immunoassays such as the enzyme
linked immunosorbant assay ("ELISA") are discussed in Maggio, E. T.
supra. Exemplary of heterogeneous assays are the radioimmunoassay,
disclosed in Yalow, et al., J. Clin. Invest. 39:1157 (1960). The
above disclosures are all incorporated herein by reference. For a
more detailed discussion of the above immunoassay techniques, see
"Enzyme-Immunoassay," by Edward T. Maggio, CRC. Press, Inc., Boca
Raton, Fla., 1980. See also, for example, U.S. Pat. Nos. 3,690,834;
3,791,932; 3,817,837; 3,850,578; 3,853,987; 3,867,517; 3,901,654;
3,935,074; 3,984,533; 3,996,345; and 4,098,876, which listing is
not intended to be exhaustive.
[0097] Heterogeneous assays usually involve one or more separation
steps and can be competitive or non-competitive. A variety of
competitive and non-competitive assay formats are disclosed in
Davalian, et al., U.S. Pat. No. 5,089,390, column 14, line 25 to
column 15, line 9, incorporated herein by reference. A typical
non-competitive assay is a sandwich assay disclosed in David, et
al., U.S. Pat. No. 4,486,530, column 8, line 6 to column 15, line
63, incorporated herein by reference.
[0098] Another binding assay approach involves the luminescent
immunoassay described in U.S. Ser. No. 07/704,569, filed May 22,
1991 entitled "Assay Method Utilizing Induced Luminescence", which
disclosure is incorporated herein by reference.
[0099] As a matter of convenience, predetermined amounts of
reagents employed in the present invention can be provided in a kit
in packaged combination. A kit can comprise in packaged combination
(a) a first oligonucleotide having the characteristic that, when
reversibly hybridized to a portion of a polynucleotide to be
detected, it is degraded under isothermal conditions by a
5'-nuclease to provide (i) a first fragment that is substantially
non-hybridizable to the polynucleotide and (ii) a second fragment
that is 3' of the first fragment and is substantially hybridizable
to the polynucleotide, (b) a second oligonucleotide having the
characteristic of at least a portion thereof hybridizing to a site
on the polynucleotide that is 3' of the site at which the first
oligonucleotide hybridizes wherein the polynucleotide is
substantially fully hybridized to such portion of the second
oligonucleotide under isothermal conditions, and (c) the above
5'-nuclease. The kit can further comprise a single nucleoside
triphosphate.
[0100] The above kits can further include members of a signal
producing system and also various buffered media, some of which may
contain one or more of the above reagents. The above kits can also
include a written description of one or more of the methods in
accordance with the present invention for detecting a
polynucleotide analyte.
[0101] The relative amounts of the various reagents in the kits can
be varied widely to provide for concentrations of the reagents
which substantially optimize the reactions that need to occur
during the present method and to further substantially optimize the
sensitivity of any assay. Under appropriate circumstances one or
more of the reagents in the kit can be provided as a dry powder,
usually lyophilized, including excipients, which on dissolution
will provide for a reagent solution having the appropriate
concentrations for performing a method or assay in accordance with
the present invention. Each reagent can be packaged in separate
containers or some reagents can be combined in one container where
cross-reactivity and shelf life permit.
EXAMPLES
[0102] The invention is demonstrated further by the following
illustrative examples. Temperatures are in degrees centigrade
(.degree. C.) and parts and percentages are by weight, unless
otherwise indicated.
Example 1
[0103] A single stranded target DNA (2.times.10.sup.8 molecules)
(M13mp19 from Gibco, BRL, Bethesda, Md.) (the "target DNA") was
combined with a 5'.sup.32P-labeled oligonucleotide probe, Probe 1,
(10 uM) (5'CGT-GGG-AAC-AAA-CGG-CGG-AT3' (SEQ ID NO: 1) synthesized
on a Pharmacia Gene Assembler (Pharmacia Biotech, Piscataway,
N.J.), an unlabeled oligonucleotide, Probe 2, (1 uM)
(5'TTC-ATC-AAC-ATT-AAA-TGT-GAG-CGA-GTA-A- CA-ACC-CGT-CGG-ATT-CTC3'
(SEQ ID NO: 2) synthesized on a Pharmacia Gene Assembler (Pharmacia
Biotech), and 7.5 units of AmpliTaq DNA polymerase (from
Perkin-Elmer Corporation, Norwalk, N.J.) in 50 uL of buffer (10 mM
Tris-HCl, pH 8.5, 50 mM KCl, 7.5 mM MgCl.sub.2, 100 uM DATP). Probe
1 was a 20-base oligonucleotide that was fully complementary to the
target DNA and had a label on the 5'-nucleotide. Probe 2, the
unlabeled probe, was designed to anneal to the target DNA 3' to,
and contiguous with, the site at which the labeled probe annealed
to the target DNA. The dATP was shown to enhance the rate of
cleavage by the polymerase. However, good results were obtained in
the absence of dATP.
[0104] The reaction mixture was incubated at 72.degree. C. and
accumulation of product, a mononucleotide, namely,
5'.sup.32P--C--OH, was determined by visualization using
autoradiography following polyacrylamide gel electrophoresis. The
fold of amplification was determined by liquid scintillation
spectrometry of excised reaction products. A 10.sup.5 fold
amplification was observed.
[0105] The above reaction protocol was repeated using, in place of
Probe 1, a labeled probe, Probe 3, (5'TCG-TGG-GAA-CAA-ACG-GCG-GAT3'
(SEQ ID NO: 3) prepared using a Pharmacia Gene Assembler) that had
21 nucleotides with one base at the 5'-end that was not
complementary, and did not hybridize with, the target DNA. The
product of this reaction was a dinucleotide, namely,
5'.sup.32P--TC--OH (SEQ ID NO: 4), that represented a 10.sup.5-fold
amplification.
[0106] The above reaction protocol was repeated with different
temperatures and different concentrations of reagents. All of the
reactions, including those mentioned above, were carried out for a
period of 3 hours. The following table summarizes the reagents and
reaction parameters and the results obtained during the
optimization procedure.
2 Probe Target Taq Fold Probe (.mu.M) number (units) Temp .degree.
C. Conditions amplification 1 1 10.sup.10 2.5 72 buffer as
described; 1.5 mM MgCl.sub.2 8.8 .times. 10.sup.2 1 10.sup.9
.vertline. .vertline. .vertline. 1.8 .times. 10.sup.3 1 10.sup.8
.dwnarw. .vertline. .vertline. N.D.* 1 10.sup.9 7.5 .vertline.
.dwnarw. 2.0 .times. 10.sup.3 1 10.sup.9 .vertline. .vertline. add
dATP(100 .mu.M) 1.4 .times. 10.sup.3 1 10.sup.8 .vertline.
.vertline. .vertline. 1.0 .times. 10.sup.4 10 10.sup.9 .vertline.
.vertline. .vertline. 1.4 .times. 10.sup.4 10 10.sup.8 .vertline.
.vertline. .dwnarw. 3.6 .times. 10.sup.4 1 10.sup.9 .vertline.
.vertline. increase MgCl.sup.2 (7.5 mM) 9.7 .times. 10.sup.3 1
10.sup.8 .vertline. .vertline. .vertline. 1.2 .times. 10.sup.4 1
10.sup.9 .vertline. .vertline. .vertline. 9.3 .times. 10.sup.3 1
10.sup.8 .vertline. .vertline. .vertline. 2.8 .times. 10.sup.4 1
10.sup.7 .vertline. .dwnarw. .vertline. N.D.* 10 10.sup.9
.vertline. 74 .vertline. 3.7 .times. 10.sup.4 10 10.sup.8
.vertline. .vertline. .vertline. 1.1 .times. 10.sup.5 10 10.sup.7
.vertline. .dwnarw. .vertline. N.D.* 3 1 10.sup.9 .vertline. 72
.vertline. 9.9 .times. 10.sup.3 1 10.sup.8 .vertline. .vertline.
.vertline. 2.6 .times. 10.sup.4 1 10.sup.7 .vertline. .dwnarw.
.vertline. N.D.* 10 10.sup.9 .vertline. 74 .vertline. 14.6 .times.
10.sup.4 10 10.sup.8 .vertline. .vertline. .vertline. 1.0 .times.
10.sup.5 10 10.sup.7 .dwnarw. .dwnarw. .dwnarw. N.D.* *N.D. = not
detected
Example 2
[0107] The reaction protocol described in Example 1 was repeated
using the following probes in place of Probe 1 or Probe 3:
[0108] Probe 4: 5'TTA-TTT-CGT-GGG-AAC-AAA-CGG-CGG-AT3' (SEQ ID NO:
5) (from Oligos Etc., Inc., Wilsonville, Oreg.). Probe 4 had 26
nucleotides with six nucleotides at its 5'-end that were not
complementary, nor hybridizable with, the target DNA. Probe 4 was
present in a concentration of 1 micromolar. The product of this
reaction was an intact seven nucleotide fragment, namely,
5'.sup.32P-TTATTTC-OH (SEQ ID NO: 6), that represented a
1.5.times.10.sup.4-fold amplification.
[0109] Probe 5:
5'GAT-TAG-GAT-TAG-GAT-TAG-TCG-TGG-GAA-CAA-ACG-GCG-GAT3' (SEQ ID NO:
7) was prepared using a Pharmacia Gene assembler and had 39
nucleotides with 19 nucleotides at its 5'-end that were not
complementary and did not hybridize with the target DNA. The
product of this reaction was an intact 20 nucleotide fragment,
namely, 5'.sup.32P-GAT-TAG-GAT-TAG-- GAT-TAG-TC-OH (SEQ ID NO: 8),
that represented a 1.5.times.10.sup.4-fold amplification.
[0110] In repeating the above reactions in the absence of Probe 2,
product was observed but the intensity of the spot on the
polyacrylamide gel was significantly less than in the presence of
Probe 2. Similar results were also observed where a 1 nucleotide
space existed between the 3'-end of Probe 2 and the second probe
when both probes were hybridized to the target DNA.
Example 3
[0111] The reaction protocol described in Example 1 was repeated
using 2.times.10.sup.9 target molecules and Probe 5 (see Example 2)
at a concentration of 1 micromolar in place of Probe 1. The
reactions were conducted for three hours at a temperature of
72.degree. C. using one of six different DNA polymerases, namely,
AmpliTaq DNA polymerase, Replitherm(TM) DNA polymerase (Epicentre),
Tfl (TM) DNA polymerase (Epicentre), Ultra Therm(TM) DNA polymerase
(Bio/Can Scientific), Thermalase Tbr(TM) DNA polymerase (Amresco)
and Panozyme(TM) DNA polymerase. The product of the reaction was a
20-nucleotide fragment (see Example 2). The following is a summary
of the results obtained.
3 Enzyme Fragment (Dicomoles) AmpliTaq 32 Replitherm 18 Tfl 5 Ultra
Therm 27 Tbr 16 Panozyme 25
[0112] The above experiments demonstrate that detectable cleavage
products were generated in a target-specific manner at a single
temperature using enzymes having 5'-nuclease activity and a labeled
oligonucleotide. The accumulation of product was enhanced by the
presence of a second oligonucleotide that was longer than the first
labeled oligonucleotide and that was annealed to the target
polynucleotide sequence 3' of the site of hybridization of the
first labeled oligonucleotide. The reactions were carried out at
temperatures very close to the melting temperature (Tm) of the
labeled oligonucleotide with the target polynucleotide
sequence.
[0113] The above discussion includes certain theories as to
mechanisms involved in the present invention. These theories should
not be construed to limit the present invention in any way, since
it has been demonstrated that the present invention achieves the
results described.
[0114] The above description and examples fully disclose the
invention including preferred embodiments thereof. Modifications of
the methods described that are obvious to those of ordinary skill
in the art such as molecular biology and related sciences are
intended to be within the scope of the following claims.
4 RUSH TRASNALATION DRAFT FROM GERMAN a) Company Board Member No.
b) Location Personally Liable (a) Day of Entry of c) Scope of
Original Partner Manag. Dir. and Signature Entry Business Capital
Manager Power of Attorney Legal Status (b) Remarks 1 2 3 4 5 6 7 1
a) Behring Diagnostics GmbH 50,000 Prof. Dr.Dr. Uwe Bicker Firm
with limited a) Oct. 24, 1996 b) Marburg DM Frankenstrasse 25
liability. Founding (signature c) The research, development, 64625
Bensheim contract Sept. 9, 1996. illegible) manufacture, and sales
of (Germany) The firm has one or b) Founding diagnostic equipment,
par- several managing di- Contract 81 ticularly diagnostic sys-
rectors. If only one 7 ff Sb tems, domestically as well managing
director, he as in foreign countries. represents the firm The firm
can conduct all exclusively. If there business transactions and are
several managing take steps directly or directors, it is re-
indirectly related to the presented by two-mana- scope of the
business. It ging directors or by can also enter into joint one
managing director ventures with the same kind and an employee with
of or similar enterprises power of attorney. domestically or in
foreign countries. 2 Dr. Friedhelm Blobel Dr. Friedhelm Blobel, a)
Feb. 26, 1997 Chemist 754 Eshbi Drive, Palo (signature 754 Eshbi
Drive Alto, CA 94301 USA illegible) Palo Alto, CA 94301 and Reiner
Gleiss, USA Reiner Gleiss Limburg, have been ap- Salesman pointed
managing Limburg (Germany) directors. 3 The address of the a) Mar
13, 1997 managing director is: b) Corrected 754 Ashby Drive, Palo
entry Alto, CA 94301 USA 4 Susan Jane Andrews, a) Apr 7, 1997 Bad
Soden; Stefan (signature Renk, Lahntal-Sterz- illegible) hausen;
Wolfgang Schneider, Steffen- berg; Gunther Veith, Marburg have been
given power of attor- ney; each of them represents the firm along
with another person having power of attorney or with a managing
director. 5 Dr. Heribert Bug, a) Apr 7, 1997 Niederweimar has
(signature been given power of illegible) attorney. He repre- sents
the firm along with another person having power of at- torney or
with a managing director. 6 Dieter Brosch, Idstein/ a) Jan 19, 1998
Kroftel has been (signature given power of at- illegible) torney.
He represents the firm along with another person having power of
attorney or with a managing di- rector. 7 a) Dade Behring Marburg
Dipl.-Engineer Gunther Power of Attorney Prof. Dr. Hwe Bicker, a)
Feb. 17, 1998 GmbH Veith, Marburg for Susan Jane An- Dr. Friedhelm
Blobel (signature c) The firm is authorized to drews, Bad Soden,
and Reiner Gleiss are illegible) conduct all commercial and Gunther
Veith, no longer managing transactions which appear Marburg has
expired. directors. necessary and useful for business purposes. Dr.
Friedhelm Blo- Dipl-Engineer Gunther b) Decision bel, Chemist, 754
Veith, Marburg, is 81. 100ff Ashby Drive, Palo managing director;
he Sb. Alto, CA 94301 USA always represents the has been given
power firm by himself, of attorney. He re- At the partners'
presents the firm meeting of January along with another 21, 1998,
the found- employee having ing contract was re- power of attorney
written and the firm or with a managing and scope of business
director were changed. The appointment of Marc C. Casper, salesman,
to managing director on Nov. 13, 1997 has been revoked as of Jan.
21, 1998. 8 Nelson J, Chai Nelson J. Chai, Sales- a) Mar 17, 1998
Salesman man, 500 Douglas Dr. (signature 500 Douglas Drive Lake
Forest, IL 60045 illegible) Lake Forest, IL 60045 has been
appointed managing director. He represents the firm along with a
managing director or another employee having power of attorney.
(Rubber Seal) (Rubber Stamp) Lowest Court Marburg It is emphasized
that the underlined entries on this photocopy have been eliminated.
(Rubber Stamp) (signature illegible) This copy represents the
entries in the official register. Additional official entries
regarding the subject do not exist. Marburg, March 20, 1998 Nr.
Grund- Vorstand a) Tag der der a) Firma oder Personilch haftende
Eintragung Ein- b) Sitz Stamm- Gesellschalter und tra- c)
Gegenstand des kapital Geschftsfuuhrer Unterschrift gung
Unternehmens DM Abwickler Prokura Rechtsverhltnisse b) Bemerkungen
1 2 3 4 5 6 7 1 a) Behring Diagnostics 50,000, Prof. Dr. Dr.
Gesellschaft mit a) 24, Okt. 1996 GmbH DM Uwe Bicker. beschrnkter
Haftung. b) Marburg FrankenstreBe 25, Gesellschaftsvertrag vom c)
Die Erforschung, Ent- 64625 Bensheim 23.9, 1996. Die wicklung,
Herstellung Gasellschaft hat einen und der Vertrieb von oder
mehrere diagnostishen Erzeug- Geschftsfuhrer. Ist nur nissen,
insbesonders ein Geschftsfuhrer such Diagnostics- bestellt,
vertritt dieser die Systemen, im In- und Gesellschaft allein.
Ausland. Sind mehrere b) Gasellsch. Die Gesellschaft kann
Geschftsfuhrer bestellt, Vertrag Bl. elle Geschfte vor- wird die
Gesellschaft 7 ff Sb nehmen und elle MaB- durch zwei nehman
argreifan, die Geschftasfuhrer mit dem Gagenstand des oder durch
einen Ge- Unternehmens Unmittel- schftsfuhrer und ber oder
mittelbar einan Prokuristen zussmmenhangen, Sie vertreten. kenn
such Beteilingungen an gleichartigen oder hnlichen Unternehmen im
In- und Ausland Uberhehmen. 2 Dr. Friedhelm Dr. Friedhelm Blobel,
a) 26, Feb. 1997 Blobel, 754 Eshbi-Drive. Chemiker, Paloalto, CA
94301, 754 Eshbi-Drive, USA und Reiner Gleiss, Paloalto CA 94301.
Limburg, sind zu Ge- USA, schftsfuhrern bestellt. Reiner Gleiss,
Kaufmann, Limburg 3 Die Anschrift des a) 13, Mrz 1997
Geschftsfuhrers Dr. Friedhelm Blobel lautet: 754 Ashby Drive Palo
Alto. CA 94301, USA. b) Berichtigend Vermarkt 4 Susan Jane Andrews,
a) 7, April 1997 Bad Soden, Stefan Renk, Lehntal-Sterzhausen,
Wolfgang Schneider. Steffenberg und Gunther Veith, Merburg ist
Prakura ertailt; jeder von ihnen vertritt die Gesellschaft mit
elnem Geschftsfuhrer odor mit sinem weiteren Prokuristen. 5 Dr.
Heribert Bug. a) 7, April 1997 Niederweimar ist Prokura ertailt; er
vertritt die Gesell- schaft mit sinem Ge- schftsfuhrer oder mit
sinem weiteren Proku- risten. 6 Dieter Brosch, a) 19, Jan. 1998
Idstein/Kroftel, ist Prokura erteilt, er vertritt die Gesell-
schaft mit sinem Ge- schftsfhrer odor mit sinem weiteren Proku-
risten. 7 a) Dade Behring Marburg Dipl. Ing. Die Prokuren fur Prof.
Dr. Uwe Bicker, a) 17, Feb. 1998 GmbH Gunther Veith, Susan Jane
Andrews, Dr. Friedhelm Blobel c) Die Geselluchaft ist Marburg Bad
Soden und und Reiner Gleiss sind berechtigt, alle Hand- Gunther
Veith. nicht mehr lungen vorzunehmen und Marburg sind
Geschftsfuhrer. alle gschaftlichen erloschen. Dr. Dipl.-Ing.
Gunther Veith, MaBnahmen zu ergreifen, Friedhelm Blobel, Marburg,
ist die zur Erfullung des Chemikor, 754 Ashby Geschftsfuhrer: er
Gesellschaftszwecks Drive, 94301 Polo vertritt die Gesellschaft
notwendig oder nutzlich Alto, Kal./USA ist stets alleine.
eracheinen. Gesamtprokurist; er Die Gesellschafter- b) Beschl. 81.
vertritt die versammlung vom 100 ff Sb. Gesellschaft 21, Januar
1998 hat den gemeinsam mit einam Gesellschaftsvertrag
Geschftsfuhrer insgesamt neu gefaBt oder einam weiteren und u.a.
die Firma und Prokuristen. den Gegenstand gendert. Die am
13.11.1997 erfolgte Bestellung des Marc N. Casper, Kaufmann, zum
Geschaftsfuhrer ist am 21.1.1998 auf- gehoben worden. 8 Nelson J.
Chai, Nelson J. Chai. a) 17, Mrz 1998 Kaufmann, Kaufmann, 500
Douglas 500 Douglas Drive, Drive, Lake Forest, IL Lake Forest,
60045, ist zum IL 60045 Geschftsfuhrer bestellt. Er vertritt
gemeinschaftlich mil einem Geschftsfuhrer oder Prokuristen. Ist er
allainiger Geschftsfuhrer, vertritt er alleine
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