U.S. patent application number 14/859078 was filed with the patent office on 2016-04-21 for primers and methods for the detection and discrimination of nucleic acids.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to IRINA NAZARENKO, AYOUB RASHTCHIAN.
Application Number | 20160108466 14/859078 |
Document ID | / |
Family ID | 26837635 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160108466 |
Kind Code |
A1 |
NAZARENKO; IRINA ; et
al. |
April 21, 2016 |
PRIMERS AND METHODS FOR THE DETECTION AND DISCRIMINATION OF NUCLEIC
ACIDS
Abstract
The present invention provides novel primers and methods for the
detection of specific nucleic acid sequences. The primers and
methods of the invention are useful in a wide variety of molecular
biology applications and are particularly useful in allele specific
PCR.
Inventors: |
NAZARENKO; IRINA;
(GAITHERSBURG, MD) ; RASHTCHIAN; AYOUB;
(GAITHERSBURG, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
CARLSBAD |
CA |
US |
|
|
Family ID: |
26837635 |
Appl. No.: |
14/859078 |
Filed: |
September 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12240914 |
Sep 29, 2008 |
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14859078 |
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09599594 |
Jun 22, 2000 |
7537886 |
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12240914 |
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60139890 |
Jun 22, 1999 |
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60175959 |
Jan 13, 2000 |
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Current U.S.
Class: |
506/9 ; 435/6.11;
435/91.2 |
Current CPC
Class: |
C12Q 2565/107 20130101;
C12Q 2525/301 20130101; C12Q 2525/301 20130101; C12Q 2563/107
20130101; C12Q 1/6858 20130101; C12Q 1/6858 20130101; C12Q 1/6816
20130101; C12Q 1/6818 20130101; C12Q 1/6832 20130101; C12Q 1/6832
20130101; C12Q 1/6827 20130101; C12Q 1/6851 20130101; C12Q 1/6827
20130101; C12Q 1/6851 20130101; C12Q 1/6818 20130101; C12Q 2525/301
20130101; C12Q 1/6816 20130101; C12Q 2563/107 20130101; C12Q
2565/107 20130101; C12Q 2563/107 20130101; C12Q 2525/301 20130101;
C12Q 2565/107 20130101; C12Q 2565/107 20130101; C12Q 2561/12
20130101; C12Q 2525/101 20130101; C12Q 2561/119 20130101; C12Q
2563/107 20130101; C12Q 2565/107 20130101; C12Q 2563/107
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A composition for quantifying or detecting one or more target
nucleic acid molecules in a sample comprising one or more
detectably labeled oligonucleotides and one or more target nucleic
acid molecules to be detected or quantified, wherein said
oligonucleotides comprise one of more detectable labels located
internally and/or at or near the 3' and/or 5' termini of said
oligonucleotides and wherein said label undergoes a detectable
change in an observable property upon becoming part of a double
stranded molecule.
2. The composition of claim 1, wherein said detectable change is an
increase or enhancement in the level of activity of the detectable
label compared to the level of activity of the detectable label in
the absence of said target nucleic acid molecules.
3. The composition of claim 2, wherein said detectable labels are
selected from the group consisting of fluorescent labels,
chemiluminescent labels and bioluminescent labels.
4. The composition of claim 3, wherein the fluorescent label is
selected from the group consisting of FAM, TAMRA, JOE, Rhodamine,
BODIPY, R6G, ROX, and EDANS.
5. The composition of claim 1, wherein said one or more detectable
labels are the same or different.
6. The composition of claim 1, wherein one or more of said
oligonucleotides comprise one or more hairpin structures.
7. The composition of claim 1, wherein one or more of said
oligonucleotides is hybridized to one or more of said nucleic acid
molecules.
8. The composition of claim 1, further comprising at least one
component selected from the group consisting of one or more
nucleotides, one or more DNA polymerases and one or more reverse
transcriptases.
9. The composition of claim 1, wherein said nucleic acid molecules
are RNA and/or DNA molecules.
10. A method for quantification or detection of one or more target
nucleic acid molecules in a sample comprising hybridizing one or
more detectably labeled oligonucleotides of claim 1 with one or
more molecules to be detected or quantified, and detecting the
presence or absence and/or quantifying the amount of said target
nucleic acid molecules.
11-17. (canceled)
18. A method for amplifying a double stranded nucleic acid
molecule, comprising: providing a first and second primer, wherein
said first primer is complementary to a sequence within or at or
near the 3'-termini of the first strand of said nucleic molecule
and said second primer is complementary to a sequence within or at
or near the 3'-termini of the second strand of said nucleic acid
molecule; hybridizing said first primer to said first strand and
said second primer to said second strand in the presence of one or
more of the polymerases, under conditions such that a third nucleic
acid molecule complementary to all or a portion of said first
strand and a fourth nucleic acid molecule complementary to all or a
portion said second strand are synthesized; denaturing said first
and third strand, and said second and fourth strands; and repeating
the above steps one or more times, wherein one or more of the
primers comprise a detectable label internally and/or at or near
its 3' and/or 5' termini and/or comprises one or more hairpin
structures.
19. The method of claim 18, wherein at least one of said primers
comprises at least one hairpin structure.
20-47. (canceled)
48. A method of determining the presence of at least one particular
nucleotide of interest at a specific position in a target nucleic
acid molecule, comprising: providing at least one target nucleic
acid molecule having said nucleotide of interest at a specific
position; contacting said target nucleic acid molecule with at
least one oligonucleotide, wherein at least a portion of the
oligonucleotide is capable of forming base pairs or hybridizing
with at least a portion of the nucleic acid molecule and wherein
the oligonucleotide comprises at least one specificity enhancing
group and/or at least one label; and contacting the oligonucleotide
and the target nucleic acid molecule with a polymerase less able to
extend the oligonucleotide when the 3'-most nucleotide of the
oligonucleotide does not base pair with the target nucleic acid and
more able to extend the oligonucleotide when the 3'-most nucleotide
of the oligonucleotide base pairs with the target nucleic acid
molecule.
49. The method of claim 48, wherein the polymerase enzyme is Tsp
DNA polymerase.
50. The method of claim 48, wherein the group is a fluorescent
moiety.
51. The method according to claim 48, wherein the groups is
attached to a nucleotide at or near the 3'-nucleotide.
52. The method according to claim 48, wherein the group is attached
to one of the ten 3'-most nucleotides.
53. The method according to claim 48, wherein the group is
detectable.
54. The method according to claim 48, wherein the oligonucleotide
is in the form of a hairpin.
55. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 12/240,914, filed Sep. 29, 2008, now
abandoned, which is a continuation of U.S. patent application Ser.
No. 09/599,594, filed Jun. 22, 2000, now U.S. Pat. No. 7,537,886,
which claims the benefit of U.S. Provisional Patent Application No.
60/139,890, filed Jun. 22, 1999, and U.S. Provisional Patent
Application No. 60/175,959, filed Jan. 13, 2000, each of which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of molecular
biology. In particular, the present invention relates to novel
primers for use in the detection and discrimination of nucleic
acids. The novel primers of the present invention will find broad
applicability in the field of molecular biology and, in particular,
in the detection of products in nucleic acid amplification
reactions and in the discrimination between alleles of a given
target gene.
[0004] 2. Related Art
[0005] Assays capable of detecting and quantifying the presence of
a particular nucleic acid molecule in a sample are of substantial
importance in forensics, medicine, epidemiology and public health,
and in the prediction and diagnosis of disease. Such assays can be
used, for example, to identify the causal agent of an infectious
disease, to predict the likelihood that an individual will suffer
from a genetic disease, to determine the purity of drinking water
or milk, or to identify tissue samples. The desire to increase the
utility and applicability of such assays is often frustrated by
assay sensitivity. Hence, it would be highly desirable to develop
more sensitive detection assays.
[0006] Nucleic acid detection assays can be predicated on any
characteristic of the nucleic acid molecule, such as its size,
sequence and, if DNA, susceptibility to digestion by restriction
endonucleases. The sensitivity of such assays may be increased by
altering the manner in which detection is reported or signaled to
the observer. Thus, for example, assay sensitivity can be increased
through the use of detectably labeled reagents. A wide variety of
such labels have been used for this purpose. Detectable labels
include, for example, radioactive isotopes, fluorescent labels,
chemiluminescent labels, bioluminescent labels and enzyme labels.
U.S. Pat. No. 4,581,333 describes the use of enzyme labels to
increase sensitivity in a detection assay. Radioisotopic labels are
disclosed in U.S. Pat. Nos. 4,358,535, and 4,446,237. Fluorescent
labels (EP 144,914), chemical labels (U.S. Pat. Nos. 4,582,789 and
4,563,417) and modified bases (EP 119,448) have also been used in
an effort to improve the efficiency with which detection can be
observed.
[0007] Although the use of highly detectable labeled reagents can
improve the sensitivity of nucleic acid detection assays, the
sensitivity of such assays remains limited by practical problems
which are largely related to non-specific reactions which increase
the background signal produced in the absence of the nucleic acid
the assay is designed to detect. In response to these problems, a
variety of detection and quantification methods using DNA
amplification have been developed.
[0008] Many current methods of identification and quantification of
nucleic acids rely on amplification and/or hybridization
techniques. While many of these involve a separation step, several
that allow detection of nucleic acids without separating the
labeled primer or probe from the reaction have been developed.
These methods have numerous advantages compared to gel-based
methods, such as gel electrophoresis, and dot-blot analysis, for
example, and require less time, permit high throughput, prevent
carryover contamination and permit quantification through real time
detection. Most of these current methods are solution-based
fluorescence methods that utilize two chromophores. These methods
utilize the phenomena of fluorescence resonance energy transfer
(FRET) in which the energy from an excited fluorescent moiety is
transferred to an acceptor molecule when the two molecules are in
close proximity to each other. This transfer prevents the excited
fluorescent moiety from releasing the energy in the form of a
photon of light thus quenching the fluorescence of the fluorescent
moiety. When the acceptor molecule is not sufficiently close, the
transfer does not occur and the excited fluorescent moiety may then
fluoresce. The major disadvantages of systems based on FRET are the
cost of requiring the presence of two modified nucleotides in a
detection oligonucleotide and the possibility that the efficiency
of the quenching may not be sufficient to provide a usable
difference in signal under a given set of assay conditions. Other
known methods which permit detection without separation are:
luminescence resonance energy transfer (LRET) where energy transfer
occurs between sensitized lanthanide metals and acceptor dyes
(Selvin, P. R., and Hearst, J. D., Proc. Natl. Acad. Sci. USA
91:10024-10028 (1994)); and color change from excimer-forming dyes
where two adjacent pyrenes can form an excimer (fluorescent dimer)
in the presence of the complementary target, resulting in a
detectably shifted fluorescence peak (Paris, P. L. et al., Nucleic
Acids Research 26:3789-3793 (1998)).
[0009] Various methods are known to those skilled in the art for
the amplification of nucleic acid molecules. In general, a nucleic
acid target molecule is used as a template for extension of an
oligonucleotide primer in a reaction catalyzed by polymerase. For
example, Panet and Khorana (J. Biol. Chem. 249:5213-5221 (1974))
demonstrate the replication of deoxyribopolynucleotide templates
bound to cellulose. Kleppe et al., (J. Mol. Biol. 56:341-361
(1971)) disclose the use of double and single-stranded DNA
molecules as templates for the synthesis of complementary DNA.
[0010] Other known nucleic acid amplification procedures include
transcription based amplification systems (Kwoh, D. et al., Proc.
Natl. Acad. Sci. USA 86:1173 (1989); PCT appl. WO 88/10315).
Schemes based on ligation ("Ligation Chain Reaction", "LCR") of two
(or more) oligonucleotides in the presence of a target nucleic acid
having a sequence complementary to the sequence of the product of
the ligation reaction have also been used (Wu, D. Y. et al.,
Genomics 4:560 (1989)). Other suitable methods for amplifying
nucleic acid based on ligation of two oligonucleotides after
annealing to complementary nucleic acids are known in the art.
[0011] PCT appl. WO 89/06700 discloses a nucleic acid sequence
amplification scheme based on the hybridization of a
promoter/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. This
scheme is not cyclic; i.e. new templates were not produced from the
resultant RNA transcripts.
[0012] EP 0 329,822 discloses an alternative amplification
procedure termed Nucleic Acid Sequence-Based Amplification (NASBA).
NASBA is a nucleic acid amplification process comprising cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA (dsDNA). The ssRNA is a first template for a
first primer oligonucleotide, which is elongated by reverse
transcriptase (RNA dependent DNA polymerase). The RNA is then
removed from resulting DNA:RNA duplex by the action of ribonuclease
H (RNase H, an RNase specific for RNA in a duplex with either DNA
or RNA). The resultant ssDNA is a second template for a second
primer. The second primer includes the sequences of an RNA
polymerase promoter (exemplified by T7 RNA polymerase) located 5'
to the primer sequence which hybridizes to the ssDNA template. This
primer is then extended by a DNA polymerase (exemplified by the
large "Klenow" fragment of E. coli DNA polymerase I), resulting in
the production of a double-stranded DNA ("dsDNA") molecule, having
a sequence identical to that of the portion of the original RNA
located between the primers and having additionally, at one end, a
promoter sequence. This promoter sequence can be used by the
appropriate RNA polymerase to make many RNA copies of the DNA.
These copies can then re-enter the cycle leading to very swift
amplification. With proper choice of enzymes, this amplification
can be done isothermally without addition of enzymes at each cycle.
Because of the cyclical nature of this process, the starting
sequence can be chosen to be in the form of either DNA or RNA.
[0013] U.S. Pat. No. 5,455,166 and EP 0 684 315 disclose a method
called Strand Displacement Amplification (SDA). This method is
performed at a single temperature and uses a combination of a
polymerase, an endonuclease and a modified nucleoside triphosphate
to amplify single-stranded fragments of the target DNA sequence. A
target sequence is fragmented, made single-stranded and hybridized
to a primer that contains a recognition site for an endonuclease.
The primer:target complex is then extended with a polymerase enzyme
using a mixture of nucleoside triphosphates, one of which is
modified. The result is a duplex molecule containing the original
target sequence and an endonuclease recognition sequence. One of
the strands making up the recognition sequence is derived from the
primer and the other is a result of the extension reaction. Since
the extension reaction was performed using a modified nucleotide,
one strand of the recognition site is modified and resistant to
endonuclease digestion. The resultant duplex molecule is then
contacted with an endonuclease which cleaves the unmodified strand
causing a nick. The nicked strand is extended by a polymerase
enzyme lacking 5'-3' exonuclease activity resulting in the
displacement of the nicked strand and the production of a new
duplex molecule. The new duplex molecule can then go through
multiple rounds of nicking and extension producing multiple copies
of the target sequence.
[0014] The most widely used method of nucleic acid amplification is
the polymerase chain reaction (PCR). A detailed description of PCR
is provided in the following references: Mullis, K. et al., Cold
Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); European Patent
(EP) 50,424; EP 84,796; EP 258,017; EP 237,362; EP 201,184; U.S.
Pat. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat. No.
4,683,194. In its simplest form, PCR involves the amplification of
a target double-stranded nucleic acid sequence. The double-stranded
sequence is denatured and an oligonucleotide primer is annealed to
each of the resultant single strands. The sequences of the primers
are selected so that they will hybridize in positions flanking the
portion of the double-stranded nucleic acid sequence to be
amplified. The oligonucleotides are extended in a reaction with a
polymerase enzyme, nucleotide triphosphates and the appropriate
cofactors resulting in the formation of two double-stranded
molecules each containing the target sequence. Each subsequent
round of denaturation, annealing and extension reactions results in
a doubling of the number of copies of the target sequence as
extension products from earlier rounds serve as templates for
subsequent replication steps. Thus, PCR provides a method for
selectively increasing the concentration of a nucleic acid molecule
having a particular sequence even when that molecule has not been
previously purified and is present only in a single copy in a
particular sample. The method can be used to amplify either single
or double-stranded nucleic acids. The essence of the method
involves the use of two oligonucleotides to serve as primers for
the template dependent, polymerase mediated replication of the
desired nucleic acid molecule.
[0015] PCR has found numerous applications in the fields of
research and diagnostics. One area in which PCR has proven useful
is the detection of single nucleotide mutations by allele specific
PCR (ASPCR) (see for example, U.S. Pat. No. 5,639,611 inventors
Wallace, et al. and U.S. Pat. No. 5,595,890 inventors Newton, et
al.). As originally described by Wu, et al. (Proceedings of the
National Academy of Sciences, USA, 86:2757-2760, 1989), ASPCR
involves the detection of a single nucleotide variation at a
specific location in a nucleic acid molecule by comparing the
amplification of the target using a primer sequence whose
3'-terminal nucleotide is complementary to a suspected variant
nucleotide to the amplification of the target using a primer in
which the 3'-terminal nucleotide is complementary to the normal
nucleotide. In the case where the variant nucleotide is present in
the target, amplification occurs more efficiently with the primer
containing the 3'-nucleotide complementary to the variant
nucleotide while in the case where the normal nucleotide is present
in the target, amplification is more efficient with the primer
containing 3'-nucleotide complementary to the normal
nucleotide.
[0016] While this technology can be used to identify single
nucleotide substitutions in a nucleic acid, it nonetheless suffers
from some drawbacks in practical applications. The difference in
efficiency of amplification between the primers may not be
sufficiently large to permit easily distinguishing between the
normal nucleotide and the mutant nucleotide. When the mismatched
primer is extended with a significant frequency in the earlier
rounds of the amplification, there may not be a large difference in
the amount of product present in the later rounds. This problem
requires careful selection of the number of amplification cycles
and reaction conditions. An additional problem with this
methodology is presented by the detection step after the
amplification. In general, this is accomplished by separating the
reaction products by electrophoresis and then visualizing the
products. The imposition of a separation step dramatically
increases the time and expense required for conducting this type of
analysis. In order to obviate the need for a separation step,
various FRET based solution phase methods of detection have been
used. These methods suffer from the drawbacks discussed above.
[0017] Whether detection of a given nucleic acid target sequence is
to be done with or without amplification of the nucleic acid sample
containing the target sequence, there remains a need in the art for
more sensitive and more discriminating methods of detecting a
target nucleic acid sequence.
[0018] Methods for detecting nucleic acid amplification products
commonly use gel electrophoresis, which separates the amplification
product from the primers on the basis of a size differential.
Alternatively amplification products can be detected by
immobilization of the product, which allows one to wash away free
primer (for example, in dot-blot analysis) and hybridization of
specific probes by traditional solid phase hybridization methods.
However, several methods for monitoring the amplification process
without prior separation of primer or probes have been described.
All of these methods are based on FRET.
[0019] One method, described in U.S. Pat. No. 5,348,853 and Wang et
al., Anal. Chem. 67:1197-1203 (1995), uses an energy transfer
system in which energy transfer occurs between two fluorophores on
the probe. In this method, detection of the amplified molecule
takes place in the amplification reaction vessel, without the need
for a separation step. The Wang et al. method uses an "energy-sink"
oligonucleotide complementary to the reverse primer. The
"energy-sink" and reverse-primer oligonucleotides have donor and
acceptor labels, respectively. Prior to amplification, the labeled
oligonucleotides form a primer duplex in which energy transfer
occurs freely. Then, asymmetric PCR is carried out to its late-log
phase before one of the target strands is significantly
overproduced.
[0020] A second method for detection of amplification product
without prior separation of primer and product is the 5' nuclease
PCR assay (also referred to as the TAQMAN.TM. assay) (Holland et
al., Proc. Natl. Acad. Sci. USA 88:7276-7280 (1991); Lee et al.,
Nucleic Acids Res. 21:3761-3766 (1993)). This assay detects the
accumulation of a specific PCR product by hybridization and
cleavage of a doubly labeled fluorogenic probe (the "TAQMAN" probe)
during the amplification reaction. The fluorogenic probe consists
of an oligonucleotide labeled with both a fluorescent reporter dye
and a quencher dye. During PCR, this probe is cleaved by the
5'-exonuclease activity of DNA polymerase if it hybridizes to the
segment being amplified. Cleavage of the probe generates an
increase in the fluorescence intensity of the reporter dye. In the
TAQMAN assay, the donor and quencher are preferably located on the
3' and 5'-ends of the probe, because the requirement that 5'-3
hydrolysis be performed between the fluorophore and quencher may be
met only when these two moieties are not too close to each other
(Lyamichev et al., Science 260:778-783 (1993)).
[0021] Another method of detecting amplification products (namely
MOLECULAR BEACONS) relies on the use of energy transfer using a
"beacon probe" described by Tyagi and Kramer (Nature Biotech.
14:303-309 (1996)). This method employs oligonucleotide
hybridization probes that can form hairpin structures. On one end
of the hybridization probe (either the 5' or 3' end) there is a
donor fluorophore, and on the other end, an acceptor moiety. In the
case of the Tyagi and Kramer method, this acceptor moiety is a
quencher, that is, the acceptor absorbs energy released by the
donor, but then does not itself fluoresce. Thus when the beacon is
in the open conformation, the fluorescence of the donor fluorophore
is detectable, whereas when the beacon is in hairpin (closed)
conformation, the fluorescence of the donor fluorophore is
quenched. When employed in PCR, the beacon probe, which hybridizes
to one of the strands of the PCR product, is in "open
conformation," and fluorescence is detected, while those that
remain unhybridized will not fluoresce. As a result, the amount of
fluorescence will increase as the amount of PCR product increases,
and thus may be used as a measure of the progress of the PCR.
[0022] Another method of detecting amplification products, which
relies on the use of energy transfer is the SUNRISE PRIMER method
of Nazarenko et al. (Nucleic Acids Research 25:2516-2521 (1997);
U.S. Pat. No. 5,866,336). SUNRISE PRIMERS are based on FRET and
other mechanisms of non-fluorescent quenching. SUNRISE PRIMERS
consist of a single stranded primer with a hairpin structure at its
5' end. The hairpin stem is labeled with a donor/quencher pair. The
signal is generated upon the unfolding and replication of the
hairpin sequence by polymerase.
[0023] While there is a body of literature on use of fluorescent
labeled nucleic acids in a variety of applications involving
nucleic acid hybridization or nucleic acid amplification, the
majority of applications involve separation of unhybridized probes
or unincorporated primers, followed by detection. None of these
methodologies, describe or discuss real time detection of probes or
primers, or changes in the fluorescence properties of a
fluorescently labeled oligonucleotide upon hybridization or
incorporation into amplified product. The surprising and novel
finding of the present invention is based, in part, on the
measurement of a change in one or more of the fluorescent
properties of labeled probes or primers upon becoming
double-stranded.
[0024] The present invention thus solves the problem of detecting
nucleic acids, in particular amplification and/or synthesis
products, by providing methods for detecting such products that are
adaptable to many methods for amplification or synthesis of nucleic
acid sequences and that greatly decrease the possibility of
carryover contamination. The compounds and methods of the invention
provide substantial improvements over those of the prior art.
First, they permit detection of the amplification or synthesis
products without prior separation of unincorporated fluorescent
labeled oligonucleotides. Second, they allow detection of the
amplification or synthesis product directly, by incorporating the
labeled oligonucleotide into the product. Third, they do not
require labeling of oligonucleotides with two different compounds
(like FRET-based methods), and thus, simplify the production of the
labeled oligonucleotides.
SUMMARY OF THE INVENTION
[0025] The present invention provides oligonucleotides that may
comprise one or more modifications internally, and/or, at or near
the 3' and/or 5' termini. Suitable modifications include, but are
not limited to, the inclusion of labels, the inclusion of
specificity enhancing groups, the inclusion of quenching moieties
and the like. The oligonucleotides of the present invention may
also comprise one or more sequences complementary to all or a
portion of a target or template sequence of interest. In some
embodiments, the oligonucleotides of the present invention may be
in the form of a hairpin. Hairpin oligonucleotides may be modified
or un-modified. Hairpin oligonucleotides of the present invention
may contain one or more single stranded regions at or near the stem
of the hairpin and may be blunt ended or comprise overhanging
sequences on the 3' and/or 5'-ends. The hairpin oligonucleotides of
the present invention may also contain any number of stem and loop
structures at any location in the oligonucleotide. In some
preferred embodiments, the oligonucleotides of the present
invention may be used for the detection and/or discrimination of
target or template nucleic acid molecules by methods involving
primer extension including, but not limited to, nucleic acid
synthesis and amplification (e.g. PCR) as well as by other methods
involving hybridization of a probe and/or primer. The
oligonucleotides of the present invention may be used with any
extension reaction known to those skilled in the art. Such
extension reactions include, but are not limited to, extension of a
primer on a DNA template using a DNA polymerase to produce a
complementary DNA strand and extension of a primer on an RNA
template using a reverse transcriptase to produce a complementary
DNA strand. The oligonucleotides of the present invention may also
be used in detection/discrimination of target or template nucleic
acid molecules using methods involving hybridization of one or more
of the oligonucleotides of the invention to one or more target
nucleic acid molecules of interest.
[0026] In one aspect, oligonucleotides of the invention may
comprise one or multiple labels (e.g. detectable labels), which may
be the same or different. In some preferred embodiments, the labels
may be fluorescent moieties. Labeled oligonucleotides of the
invention may be used to detect the presence or absence of or to
quantify the amount of nucleic acid molecules in a sample by, for
example, hybridization of such oligonucleotides to such nucleic
acid molecules. Optionally, such oligonucleotides may be extended
in a synthesis and/or amplification reaction and
detection/quantification may be accomplished during or after such
reactions. In accordance with one aspect of the invention, such
detection/quantification is based on the observation that the
labeled oligonucleotides in double-stranded form have a detectable
change in one or more properties (preferably a fluorescent
property) compared to the oligonucleotides in single-stranded form.
In another aspect of the invention, a change in a detectable
property (preferably a fluorescent property) upon extension of the
oligonucleotide of the invention is used to detect/quantify a
target/template nucleic acid. Fluorescent properties in which a
change may be detected include, but are not limited to, fluorescent
intensity (increase or decrease), fluorescent polarization,
fluorescence lifetime and quantum yield of fluorescence. Thus,
hybridization and/or extension of the labeled oligonucleotides of
the invention to a nucleic acid molecule to be detected/quantified
results in a detectable change in one or more of the labels used
and, in particular, when using fluorescent labels, a detectable
change in one or more fluorescent properties. In this aspect of the
invention, multiple different oligonucleotides may be used to
detect multiple different target sequences in the same sample (e.g.
multiplexing) and such different oligonucleotides may be
differentially labeled to allow simultaneous and/or sequential
detection of the multiple target sequences.
[0027] In another aspect, the present invention provides modified
oligonucleotides comprising one or more specificity enhancing
groups. In some preferred embodiments, oligonucleotides of the
present invention may be provided with one or more specificity
enhancing groups that render such oligonucleotides substantially
less extendable, for example in a synthesis or amplification
reaction, when the 3'-most nucleotide of the oligonucleotide is not
base paired with a target or template nucleic acid sequence. In
some embodiments, the specificity enhancing group may be placed at
or near the 3'most nucleotide of the oligonucleotide. The
specificity enhancing group may be attached to the oligonucleotide
using any methodology known to those of skill in the art and may be
attached to the oligonucleotide via a linker group. Such linker
groups may be of varying length and chemical composition, i.e.,
hydrophobicity, charge etc. The specificity enhancing groups of the
present invention may be attached to any part of the nucleotide to
be modified, i.e., base, sugar or phosphate group. Specificity
enhancing groups of the present invention may be or include
detectable groups, including but not limited to, fluorescent
groups, chemiluminescent, radiolabeled groups and the like. In some
embodiments, the specificity enhancing groups of the present
invention may be fluorescent groups which undergo a detectable
change in one or more fluorescent properties upon extension of the
oligonucleotide or may be any other detectable label allowing
detection of the nucleic acid of interest. Preferably, the label
exhibits a detectable change when the oligonucleotide of the
invention is extended in a synthesis or amplification reaction.
[0028] Oligonucleotides of the present invention may be in the form
of a hairpin. The hairpins of the present invention preferably
comprise at least one stem structure and at least one loop
structure. The sequences which form the stem structure by base
pairing may be of any length and preferably contain at least a
portion of a sequence complementary to a target or template
sequence. For example, the sequence of an oligonucleotide may be
selected so as to form a hairpin structure at a temperature below
the temperatures used in a synthesis or amplification reaction by
first selecting a sequence at least partially complementary to a
portion of a nucleic acid target or template sequence and then
adding one or more nucleotides to the 5'-end of the oligonucleotide
that are complementary to the nucleotides at the 3'-end of the
oligonucleotide. At a reduced temperature, the complementary
nucleotides at the 3' and 5' ends can base pair forming a stem
structure. The number of complementary nucleotides to be added may
be selected by determining the desired melting temperature of the
stem structure. The melting temperature preferably is high enough
that the oligonucleotide is in the hairpin structure when the
reaction mixture is being prepared thereby preventing the
oligonucleotide from mis-annealing to the target or template
nucleic acid molecule but low enough such that all or portion of
the oligonucleotides are capable of assuming a linear structure and
annealing to the target or template at the appropriate point in the
synthesis or amplification reaction. The selection of an
appropriate melting temperature for the stem structure is routine
for those of ordinary skill in the art.
[0029] The oligonucleotides of the present invention may
incorporate more than one of the characteristics described above or
combinations thereof. For example, an oligonucleotide may comprise
one or more labels and/or one or more specificity enhancing groups
and/or one or more hairpin structures.
[0030] In another aspect, one or more of the oligonucleotides of
the present invention may be covalently or non-covalently attached
to a support by any means known to those skilled in the art. Such
support bound oligonucleotides may be used to carry out the methods
of the present invention. For example, the detection or
quantification of nucleic acid molecules may be accomplished on a
support and/or the synthesis or amplification of nucleic acids may
be accomplished on a support. Such a support may be solid or
semisolid and may be made of any material known to those skilled in
the art.
[0031] In one aspect, the present invention provides for reaction
mixtures or compositions for use in a process for the synthesis
and/or amplification of one or more nucleic acid molecules
complementary to all or a portion of one or more nucleic acid
target or template molecules of interest. In some preferred
embodiments, the reaction mixture may comprise at least a first and
preferably a first and a second oligonucleotide primer of the
invention which primers may be the same or different and may
contain the same or different labels and/or specificity enhancing
groups. Such first primer preferably comprises at least one
sequence which is at least partially complementary to said target
or template nucleic acid and which primes synthesis of a first
extension product that is complementary to all or a portion of said
target or template nucleic acid. Such second oligonucleotide primer
preferably comprise a sequence which is at least partially
complementary to all or a portion of said first extension product
and primes the synthesis of a second extension product which is at
least partially complementary to all or a portion of said first
extension product. In some embodiments, the reaction mixture may
comprise one or more oligonucleotide primers of the invention,
which may be the same or different, and which may contain one or
more of the same or different labels and/or specificity enhancing
groups. For example, the reaction mixture or composition may
comprise more than one oligonucleotide primer, wherein at least one
of said primers is in the form of a hairpin and another is not. In
another aspect, one primer may be provided with a label that
undergoes a detectable change in one or more properties upon
hybridization and/or extension while a second primer may be in the
form of a hairpin and/or comprise a specificity enhancing group. In
another aspect, both the first and the second primer may be in the
form of a hairpin and may also comprise labels and/or specificity
enhancing groups as described above. Such reaction mixtures or
compositions of the present invention may further comprise one or
more components selected from a group consisting of one or more
nucleotides, one or more DNA polymerases, one or more reverse
transcriptases, one or more buffers or buffering salts, one or more
target or template molecules and one or more products produced by a
synthesis/amplification reaction of the present invention. Thus,
the invention relates generally to compositions/reaction mixtures
produced to carry out the invention and/or to composition/reaction
mixtures resulting from carrying out the invention.
[0032] The present invention relates to a method for detecting the
presence or absence of a nucleic acid molecule or for quantifying
the amount of a nucleic acid molecule in a sample comprising:
[0033] (a) contacting a sample thought to contain one or more
nucleic acid molecules with one or more oligonucleotides of the
invention; and [0034] (b) detecting the presence or absence or
quantifying the amount of nucleic acid molecules in said sample. In
some embodiments, the oligonucleotide may be labeled and the
detecting step may involve the detection of a change in one or more
fluorescent or other detectable properties of a the labeled
oligonucleotide of the present invention. In some embodiments, the
fluorescent property which undergoes a change is the intensity of
fluorescence. In some embodiments, an increase in fluorescence
intensity is detected.
[0035] Preferably the oligonucleotides of the invention are
incubated under conditions sufficient to allow hybridization of
such oligonucleotides to the nucleic acid molecules in the sample.
In a preferred aspect, the detection or quantification step
includes a comparison of a control sample (without nucleic acid
molecules present) to the sample containing nucleic acid molecules.
Additional control samples containing known amounts of nucleic acid
molecules may be used in accordance with the invention as a
positive control for comparison purposes to determine the exact or
approximate amount of the nucleic acid molecules present in the
unknown sample.
[0036] In a related aspect, the invention relates to detection or
quantification of nucleic acid molecules in a sample during or
after nucleic acid synthesis or amplification. Thus, the invention
relates to a method for detection or quantification of one or more
nucleic acid molecules in a sample comprising: [0037] (a) mixing
one or more nucleic acid templates or target nucleic acid molecules
of the sample with one or more oligonucleotides for the invention;
[0038] (b) incubating said mixture under conditions sufficient to
synthesize or amplify one or more nucleic acid molecules
complementary to all or a portion of said templates or target
molecules, wherein said synthesized or amplified nucleic acid
molecules comprise said oligonucleotide; and [0039] (c) detecting
or quantifying said synthesized or amplified nucleic acid
molecules. In some embodiments, the oligonucleotide may be labeled
and the detecting step may involve the detection of a change in one
or more fluorescent or other detectable properties of the labeled
oligonucleotide of the present invention. In some embodiments, the
fluorescent property which undergoes a change is the intensity of
fluorescence. In some embodiments, an increase in fluorescence
intensity is detected.
[0040] Conditions sufficient to synthesize or amplify one or more
nucleic acid molecules complementary to all or a portion of said
templates or target molecules preferably comprise incubating the
template/oligonucleotide mixture in the presence of one or more
nucleotides and one or more polymerases and/or reverse
transcriptases (preferably DNA polymerases and most preferably
thermostable DNA polymerases). In a most preferred aspect, the
amplification process used is polymerase chain reaction (PCR) or RT
PCR, although other amplification methods may be used in accordance
with the invention. In this aspect of the invention, the
detection/quantification step may be accomplished during
amplification or synthesis or after synthesis or amplification is
complete. For detection during an amplification reaction, a
thermocycler capable of real time fluorescence detection may be
used. Further, the nucleic acid synthesis or amplification method
preferably produces double-stranded nucleic acid molecules
(preferably double-stranded DNA/DNA or DNA/RNA molecules) and the
presence or absence or amount of such double-stranded molecules may
be determined by this method of the invention. In a preferred
aspect, using the labeled oligonucleotides of the invention as a
primer during synthesis or amplification, the labeled
oligonucleotide primer is incorporated into the synthesized or
amplified molecule thereby creating a labeled product molecule
(which may be single-stranded or double-stranded). In another
aspect, the synthesized or amplified nucleic acid molecules
produced in accordance with the invention may contain one or more
labels, which may be the same or different. In a preferred aspect,
the detection or quantification step includes a comparison of a
control sample to the sample containing the target/template nucleic
acid molecules of interest. Additional control samples containing
known amounts of target/template may be used as a positive control
for comparison purposes and/or to determine the exact or
approximate amount of target/template in an unknown sample.
[0041] More specifically, the invention is directed to a method for
amplifying a double-stranded nucleic acid target molecule (e.g.,
DNA/DNA; RNA/RNA; or RNA/DNA), comprising: [0042] (a) providing at
least a first and a second primer, wherein said first primer is
complementary to a sequence within or at or near the 3'-termini of
a first strand of said nucleic molecule and said second primer is
complementary to a sequence within or at or near the 3'-termini of
the second strand of said nucleic acid molecule; [0043] (b)
hybridizing said first primer to said first strand and said second
primer to said second strand in the presence of one or more of
polymerases or reverse transcriptases, under conditions such that a
third nucleic acid molecule complementary to all or a portion of
said first strand and a fourth nucleic acid molecule complementary
to all or a portion of said second strand are synthesized; [0044]
(c) denaturing said first and third strand, and said second and
fourth strands; and [0045] (d) repeating steps (a) to (c) one or
more times, wherein one or more of said primers are
oligonucleotides of the present invention. In some embodiments, at
least one of the primers comprises a label that undergoes a
detectable change in one or more fluorescent or other detectable
properties upon hybridization and/or extension. In some
embodiments, at least one of the primers comprises a specificity
enhancing group that renders the primer substantially less
extendable when the 3'-nucleotide of the primer is not base paired
with the target molecule. In some embodiments, one or more of the
primers is in the form of a hairpin. In some embodiments, at least
one of the primers is in the form of a hairpin and further
comprises a detectable label and/or a specificity enhancing
group.
[0046] In a further aspect, the present invention provides a method
for the direct detection of amplification or synthesis products in
which the detection may be performed without opening the reaction
tube. This embodiment, the "closed-tube" format, reduces greatly
the possibility of carryover contamination with amplification or
synthesis products. The closed-tube method also provides for high
throughput analysis of samples and may be automated. The
closed-tube format significantly simplifies the detection process,
eliminating the need for post-amplification or post-synthesis
analysis such as gel electrophoresis or dot-blot analysis.
[0047] In another aspect, the invention relates to a method for
hybridizing or binding one or more of the oligonucleotides of the
invention with one or more nucleic acid molecules of interest
comprising: [0048] (a) mixing one or more of said oligonucleotides
with one or more of said nucleic acid molecules; and [0049] (b)
incubating said mixture under conditions sufficient to hybridize or
bind one or more of said oligonucleotides with one or more of said
nucleic acid molecules. In a preferred aspect, at least one or more
of the oligonucleotides used in this method are hairpins and more
preferably, the one or more oligonucleotides are hairpin molecules
comprising one or more specificity enhancing groups and/or one or
more labels.
[0050] The invention also relates to methods of synthesis or
amplification of one or more nucleic acid molecules comprising:
[0051] (a) mixing one or more templates or target nucleic acid
molecules with one or more oligonucleotides of the invention; and
[0052] (b) incubating said mixture under conditions sufficient to
synthesize or amplify one or more nucleic acid molecules
complementary to all or a portion of said templates or target
molecules. In a preferred aspect, the oligonucleotides are hairpins
and more preferably are hairpin molecules comprising one or more
specificity enhancing groups and/or one or more labels. Conditions
sufficient to synthesize or amplify one or more nucleic acid
molecules complementary to all or a portion of said templates or
target molecules preferably comprise incubating the
templates/oligonucleotide mixture (e.g., the
template-oligonucleotide complex) in the presence of one or more
nucleotides and one or more polymerases and/or one or more reverse
transcriptases (preferably DNA polymerases and most preferably
thermostable DNA polymerases). In a most preferred aspect, the
amplification process used is polymerase chain reaction (PCR) or RT
PCR, although other amplification methods may be used in accordance
with the invention. Further, the nucleic acid synthesis or
amplification methods preferably produces double stranded nucleic
acid molecules (preferably double stranded DNA/DNA or DNA/RNA
molecules). Use of the oligonucleotides of the invention allows for
more efficient synthesis and/or amplification of nucleic acid
molecules.
[0053] More specifically, the invention is directed to a method for
amplifying a double stranded nucleic acid target molecules
comprising: [0054] (a) providing a first and second primer, wherein
said first primer is complementary to a sequence within or at or
near the 3' termini of the first strand of said nucleic acid
molecule and said second primer is complementary to a sequence
within or at or near the 3' termini of the second strand of said
nucleic acid molecule; [0055] (b) hybridizing said first primer to
said first strand and said second primer to said second strand in
the presence of one or more polymerases or reverse transcriptases,
under conditions such that a third nucleic acid molecule
complementary to all or a portion of said first strand and a fourth
nucleic acid molecule complementary to all or a portion of said
second strand are synthesized; [0056] (c) denaturing said first and
third strands, and said second and first strands; and [0057] (d)
repeating steps (a) to (c) one or more times, wherein one or more
of said primers are oligonucleotides of the present invention. In
one embodiment, the oligonucleotides of the invention used are
hairpins, and preferably are hairpins comprising one or more
specificity enhancing groups and/or one or more labels.
[0058] The invention also provides the embodiments of the above
methods wherein the nucleic acid molecule to be
detected/quantified/amplified/synthesized is an RNA or a DNA
molecule, and wherein such molecule is either single-stranded or
double-stranded.
[0059] The invention also provides the embodiments of the above
methods wherein one or a number of the primers or oligonucleotides
of the present invention comprise at least one nucleotide
derivative. Examples of such derivatives include, but are not
limited to, a deoxyinosine residue, a thionucleotide, a peptide
nucleic acid and the like.
[0060] The invention also provides the embodiment of the above
methods wherein the nucleic acid target or template molecule is
polyadenylated at its 3' end (e.g., poly(A) RNA or mRNA), and/or at
least one of the primers or oligonucleotides of the invention
contains a poly(T) sequence, and/or at least one of the other of
the primers or oligonucleotides of the invention contains at least
one deoxyinosine residue. In a related aspect, the template or
target nucleic acid is an mRNA molecule, at least one
primer/oligonucleotide is labeled and comprises a poly(T) sequence
and at least one primer/oligonucleotide comprises at least one
deoxyinosine residue.
[0061] As will be further appreciated, the labeled oligonucleotide
sequences of the invention may be employed in other amplification
methods, such as those involving the application of PCR to the
amplification of cDNA-ends derived from mRNAs using a single gene
specific primer. Thus, labeled oligonucleotides of the invention
can be used in methods such as "RT-PCR," "5'-RACE," "anchor PCR"
and "one-sided PCR," which facilitate the capture of sequence from
5'-ends of mRNA. The methods of the invention are adaptable to many
methods for amplification of nucleic acid sequences, including PCR,
LCR, SDA and NASBA, and other amplification systems known to those
of ordinary skill in the art.
[0062] In another aspect of the invention, the invention is
directed to a method for determining the activity or amount of a
polymerase in a sample, comprising amplifying a nucleic acid
molecule, comprising: [0063] (a) providing a first and second
primer, wherein said first primer is complementary to a sequence
within or at or near the 3'-termini of the first strand of said
nucleic acid molecule and said second primer is complementary to a
sequence within or at or near the 3'-termini of the second strand
of said nucleic acid molecule; [0064] (b) hybridizing said first
primer to said first strand and said second primer to said second
strand in the presence of said polymerase, under conditions such
that a third nucleic acid molecule complementary to all or a
portion of said first strand and a fourth nucleic acid molecule
complementary to all or a portion of said second strand are
synthesized; [0065] (c) denaturing said first and third strand, and
said second and fourth strands; and [0066] (d) repeating steps (a)
to (c) one or more times; and [0067] (e) detecting the
amplification product, wherein at least one of the primers are
oligonucleotides of the present invention, and wherein the amount
of the amplification product produced is indicative of the activity
or amount of the polymerase.
[0068] In some embodiments, the amount of the amplification product
produced is determined by detecting a change in one or more
fluorescent or other detectable properties of an incorporated
detectable label.
[0069] Generally, the invention thus relates to a method for
determining the activity or the amount of polymerase or reverse
transcriptase in a sample comprising: [0070] (a) mixing a sample
thought to contain a polymerase or reverse transcriptase with one
or more nucleic acid templates and one or more labeled
oligonucleotides of the invention; [0071] (b) incubating said
mixture under conditions sufficient to allow synthesis or
amplification of one or more nucleic acid molecules complementary
to all or a portion of said templates, wherein said synthesized or
amplified nucleic acid molecules comprise said oligonucleotides;
and [0072] (c) determining the activity or amount of said
polymerase or reverse transcriptase in said sample based on
detection of one or more detectable labels.
[0073] In another aspect, the invention relates to quenching
background fluorescence during detection of nucleic acid molecules
or polymerases in accordance with the methods of the invention. In
this aspect of the invention, one or more quenching agents which
bind one or more labeled single-stranded nucleic acid molecules are
used to quench the fluorescence produced by such single-stranded
molecules. In a preferred aspect, the quenching agent is specific
for single-stranded molecules and will not substantially interact
with double-stranded labeled nucleic acid molecules. Thus,
fluorescently labeled oligonucleotides of the invention will be
quenched or substantially quenched in the presence of such agents.
Upon interaction with the target molecule or during amplification
or synthesis reactions, the double-stranded nucleic acid molecule
formed which comprise the fluorescently labeled oligonucleotides of
the invention will not substantially interact with such agents and
thus will not be quenched by such agents. This aspect of the
invention thus allows for reduced background fluorescence and
enhanced detection of target nucleic acid molecules in the methods
of the invention. Preferred quenchers for use in the invention
include one or more single-stranded binding proteins. In another
aspect, such quenching agents may include blocking oligonucleotides
which contain one or more quenchers, for example, DABCYL. In
another aspect, the quenching moiety may be part of the
oligonucleotide of the invention. For example, one or more
quenching moieties may be incorporated into one or more stem
structures of the hairpin of the invention. Such stem structures
may also incorporate one or more labels and in the hairpin
configuration, the quenching moieties reduce the level of
background activity of the label. Upon denaturation (unfolding) of
the stem structure, the quenching of the label is reduced or
prevented.
[0074] In another embodiment, the invention relates to a
composition comprising one or more labeled oligonucleotide of the
invention, wherein the label is a detectable label, and wherein the
oligonucleotide is selected from the group consisting of DNA and
RNA. The labeled oligonucleotides of the invention may be primers
and/or probes, depending on the use. The compositions of the
invention may further comprise one or more components selected from
the group consisting of one or more polymerases, one or more
quenching agents, one or more nucleotides, one or more nucleic acid
molecules (which may be templates or nucleic acid molecules which
may comprise one or more oligonucleotides of the invention), and
one or more buffering salts.
[0075] In another embodiment of the invention, the label is a
member of a FRET pair. In this embodiment, one or more labeled
oligonucleotides of the invention containing a single or multiple
members of a FRET pair internally, and/or, at or near the 3' and/or
5' end. In a preferred aspect, the labeled moiety is one or more
fluorescent moieties whose emission may then be measured to assess
the progress of the reaction.
[0076] The present invention also relates to kits for the detection
or measurement of nucleic acid synthesis or amplification products
or for the measurement or detection of nucleic acid molecules of
the invention. Such kits may be diagnostic kits where the presence
of the nucleic acid being amplified or synthesized is correlated
with the presence or absence of a disease or disorder. Kits of the
invention may also be used to detect or determine activity or
amount of a polymerase in a sample. In addition, kits of the
invention may be used to carry out synthesis, amplification or
other extension reactions using the oligonucleotides of the
invention. Preferred kits of the invention may comprise one or more
containers (such as vials, tubes, and the like) configured to
contain the reagents used in the methods of the invention and
optionally may contain instructions or protocols for using such
reagents. The kits of the invention may comprise one or more
components selected from the group consisting of one or more
oligonucleotides of the invention (including probes and/or
primers), one or more DNA polymerases, such as a thermostable
polymerase, one or more reverse transcriptases, or any other DNA or
RNA polymerase, one or more agents capable of quenching one or more
of the labels, one or more buffers or buffering salts, one or more
nucleotides, one or more target/template molecules (which may used
for determining reaction performance, i.e., control reactions) and
other reagents for analysis or further manipulation of the products
or intermediates produced by the methods of the invention. Such
additional components may include components used for cloning
and/or sequencing and components or equipment needed for the
detection or quantification of the nucleic acid molecule of
interest.
[0077] The invention also relates to any of the products or
intermediates (e.g, nucleic acid molecules) produced by carrying
out the methods of the invention. The invention also relates to
vectors or host cells containing such products or intermediates
produced by the methods of the invention. Introduction of such
vectors into host cells may be accomplished using any of the
cloning and transformation techniques known to those skilled in the
art.
BRIEF DESCRIPTION OF THE FIGURES
[0078] FIGS. 1A-1B are schematic representations of the
homogeneous/real-time detection system of the invention. A change
in one or more fluorescent or other detectable properties can be
detected either through the incorporation of the labeled primer
into the double-stranded amplification product (FIG. 1A), or
through the direct hybridization of the labeled probe to the
nucleic acid target (FIG. 1B). In accordance with the invention,
the nucleic acid molecules detected or quantified can be a
synthesized or amplified product or a nucleic acid molecule found
in nature. Such nucleic acid molecules may be single or double
stranded and can be RNA, DNA or RNA/DNA hybrids. In accordance with
the invention, any one or more labels (which may be the same or
different) may be used.
[0079] FIGS. 2A-2B are graphs of fluorescent intensity as a
function of temperature which shows the effect of hybridization on
the fluorescence of internally (FIG. 2A) and 5'-fluorescein labeled
(FIG. 2B) oligonucleotides. Labeled oligonucleotides were tested
for fluorescence under different temperatures. Single-stranded (SS)
or double-stranded (DS) oligonucleotides were melted as described
in Example 4. For 5'-labeled oligonucleotides, conversion from SS
oligonucleotides to DS oligonucleotides caused a decrease in
fluorescence, while for internally labeled oligonucleotides,
conversion from SS oligonucleotides to DS caused an increase in
fluorescence.
[0080] FIG. 3 is a graph of fluorescent intensity as a function of
wavelength which shows fluorescence of 3'-TAMRA oligonucleotide in
the presence of complementary and non-complementary
oligonucleotides. In presence of complement (to create a double
stranded molecule), the fluorescence increased compared to the
single stranded form (see Example 5).
[0081] FIG. 4 is a graph of fluorescence as a function of
wavelength which shows the effect of hybridization on the
fluorescence of oligonucleotides 5'-labeled with fluorescein and
BODIPY 530/550. In the presence of the complement oligonucleotide
(to create a double stranded molecule), the fluorescence increased
in case of BODIPY dye and decreased in case of fluorescein.
[0082] FIG. 5A is a graph of fluorescent intensity as a function of
the number of cycles of amplification performed which shows
quantitative PCR of IL4 cDNA with an internally labeled primer
(Panel A). PCR was performed as described in Example 7. Data from
ABI PRIZM.TM.7700 Sequence Detector were treated according to the
manufacturer's instructions with minor modifications (FIG. 5B).
FIG. 5C is a standard curve plotting the number of cycles of
amplification against the starting quantity of template DNA.
[0083] FIG. 6 is a graph of fluorescent intensity as a function of
the number of cycles of amplification performed which shows IL4
cDNA PCR with a primer post-synthetically labeled with fluorescein.
PCR was performed as described in Example 8. Real-time
amplification data were exported from ABI PRIZMT.TM. 7700 Sequence
Detector in Excel.
[0084] FIG. 7 is a graph of fluorescent intensity as a function of
the number of cycles of amplification performed which shows
detection of b-actin cDNA by PCR with a primer internally labeled
with fluorescein. PCR was performed as described in Example 9.
[0085] FIG. 8 is a graph of fluorescent intensity as a function of
the number of cycles of amplification performed which shows b-Actin
cDNA PCR with a primer internally labeled through a 5'-detection
tail. PCR was performed as described in Example 10.
[0086] FIG. 9 is a schematic representation of allele specific
PCR.
[0087] FIG. 10 is a photograph of an agarose gel showing the
results of an allele specific PCR reaction comparing the primers of
the present invention to standard primers.
[0088] FIG. 11 is a plot of fluorescence as a function of the
number of cycles of PCR performed in an allele specific PCR
reaction comparing the hairpin primers of the present invention to
standard linear primers.
[0089] FIG. 12 is a plot of fluorescence as a function of the
number of cycles of PCR performed in an allele specific PCR
reaction comparing the hairpin primers of the present invention to
standard linear primers using a two step PCR reaction format.
[0090] FIG. 13A shows a bar graph of the fluorescence intensity
obtained at the end point of an allele specific PCR reaction using
the primers of the present invention. FIG. 13B is a photograph of
the PCR tubes in which the allele specific reaction was conducted
illuminated with ultraviolet light.
[0091] FIG. 14 is a photograph of an agarose gel showing the
effects of target DNA concentration on an allele specific PCR
reaction using the primers of the present invention.
[0092] FIG. 15 is a photograph of an agarose gel showing the
results of an allele specific reaction comparing the results
obtained using Tsp DNA polymerase to Taq DNA polymerase using
standard primers.
[0093] FIGS. 16A-16B are photographs of an ethidium bromide stained
agarose gel showing the results of comparison of the hairpin
oligonucleotides of the present invention to linear
oligonucleotides in an amplification reaction using varying amounts
of template DNA. FIG. 16A shows the amplification of a 3.6. kb
fragment of the human beta-globin gene using a first primer set.
FIG. 16B shows the amplification of a 3.6 kb fragment of the human
beta-globin gene using a second primer set.
[0094] FIG. 17 is a photograph of an ethidium bromide stained
agarose gel showing the results of comparison of the hairpin
oligonucleotides of the present invention to linear
oligonucleotides in an amplification reaction to produce varying
sized amplification products. Panel A shows the amplification of a
1.3 kb fragment of the NF2 gene. Panel B shows the amplification of
a 1.6 kb fragment of the NF2 gene.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and Abbreviations
[0095] In the description that follows, a number of terms used in
recombinant DNA technology are extensively utilized. As used
herein, the following terms shall have the abbreviations
indicated:
[0096] ASP, allele-specific polymerase chain reaction
[0097] bp, base pairs
[0098] DAB or DABCYL, 4-(4'-dimethylaminophenylazo)benzoic acid
[0099] EDANS, 5-(2'-aminoethyl)aminonapthalene-1-sulfonic acid
[0100] FAM or Flu, 5-carboxyfluorescein
[0101] JOE, 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein
[0102] HPLC, high-performance liquid chromatography
[0103] NASBA, nucleic acid sequence-based amplification
[0104] Rhod, rhodamine
[0105] ROX, 6-carboxy-X-rhodamine
[0106] R6G, 6-carboxyrhodamine
[0107] TAMRA, N,N,N',N'-tetramethyl-6-carboxyrhodamine
[0108] Amplification. As used herein, "amplification" refers to any
in vitro method for increasing the number of copies of a nucleotide
sequence with the use of a polymerase. Nucleic acid amplification
results in the incorporation of nucleotides into a nucleic acid
(e.g., DNA) molecule or primer thereby forming a new nucleic acid
molecule complementary to the nucleic acid template. The formed
nucleic acid molecule and its template can be used as templates to
synthesize additional nucleic acid molecules. As used herein, one
amplification reaction may consist of many rounds of nucleic acid
synthesis. Amplification reactions include, for example, polymerase
chain reactions (PCR). One PCR reaction may consist of 5 to 100
"cycles" of denaturation and synthesis of a nucleic acid
molecule.
[0109] Specificity enhancing group. As used herein "specificity
enhancing group" refers to any molecule or group of molecules that
causes an oligonucleotide of the present invention to be
substantially less extendable when the 3'-most nucleotide of the
oligonucleotide is substantially not base paired with a nucleotide
on the nucleic acid target/template molecule. Any type of group may
be used. Preferred examples include, but are not limited to,
fluorescent groups, modified nucleotides, small molecules, haptens
and the like. Specificity enhancing groups may be attached at any
position of the oligonucleotide so long as they make the
oligonucleotide substantially less extendable when the 3'-terminal
nucleotide of the oligonucleotide is substantially not base paired
with the corresponding nucleotide of the target/template nucleic
acid. Such groups are preferably attached to the primer at or near
the 3'-end of the primer but may be attached at other positions as
well. Preferably, they are attached to one or more of the 25 bases
adjacent to the 3'-end of the primer. In some preferred
embodiments, such groups may be attached to one or more of the 20
bases adjacent to the 3'-end of the oligonucleotide, or to the 15
bases adjacent to the 3'-end or to the 10 base pairs adjacent to
the 3'-end or, most preferably to one or more of the five bases
adjacent to the 3'-end of the oligonucleotide. In addition,
specificity enhancing groups may be attached to the 3'-most
nucleotide so long as the presence of the group does not prevent or
inhibit the extension of the primer when the 3'-most nucleotide of
the primer is complementary to the corresponding nucleotide on the
target/template molecule more than the extension is inhibited when
the 3'-most nucleotide is substantially not base paired to the
target/template. Any group that can decrease the stability of the
duplex formed by the primer and template when the 3'-most
nucleotide of the primer is not complementary the corresponding
nucleotide of the target/template and/or any group that can make a
polymerase less efficient at extending the 3'-end of the
oligonucleotide when the 3'-most nucleotide is not complementary to
the corresponding nucleotide of the template/target may be used to
practice the present invention. In some embodiments, the
specificity enhancing groups of the invention may be modified
nucleotides incorporated into the sequence of the primer. Such
modifications may be made at the base, sugar or phosphate portion
of the nucleotide and include but are not limited to phophothioate
nucleotides, phosphonate nucleotides, peptide nucleic acids and the
like.
[0110] Polymerase. As used herein "polymerase" refers to any enzyme
having a nucleotide polymerizing activity. Polymerases (including
DNA polymerases and RNA polymerases) useful in accordance with the
present invention include, but are not limited to, Thermus
thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA
polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga
maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or
VENT.TM.) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase,
DEEPVENT.TM. DNA polymerase, Pyrococcus woosii (Pwo) DNA
polymerase, Bacillus sterothermophilus (Bst) DNA polymerase,
Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus
acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac)
DNA polymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermus
ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME.TM.) DNA
polymerase, Methanobacterium thermoautotrophicum (Mth) DNA
polymerase, mycobacterium DNA polymerase (Mtb, Mlep), and mutants,
and variants and derivatives thereof. RNA polymerases such as T3,
T5 and SP6 and mutants, variants and derivatives thereof may also
be used in accordance with the invention. Generally, any type I DNA
polymerase may be used in accordance with the invention although
other DNA polymerases may be used including, but not limited to,
type III or family A, B, C etc. DNA polymerases.
[0111] Polymerases used in accordance with the invention may be any
enzyme that can synthesize a nucleic acid molecule from a nucleic
acid template, typically in the 5' to 3' direction. The nucleic
acid polymerases used in the present invention may be mesophilic or
thermophilic, and are preferably thermophilic. Preferred mesophilic
DNA polymerases include T7 DNA polymerase, T5 DNA polymerase,
Klenow fragment DNA polymerase, DNA polymerase III and the like.
Preferred thermostable DNA polymerases that may be used in the
methods of the invention include Taq, Tne, Tma, Pfu, Tfl, Tth,
Stoffel fragment, VENT.TM. and DEEPVENT.TM. DNA polymerases, and
mutants, variants and derivatives thereof (U.S. Pat. No. 5,436,149;
U.S. Pat. No. 4,889,818; U.S. Pat. No. 4,965,188; U.S. Pat. No.
5,079,352; U.S. Pat. No. 5,614,365; U.S. Pat. No. 5,374,553; U.S.
Pat. No. 5,270,179; U.S. Pat. No. 5,047,342; U.S. Pat. No.
5,512,462; WO 92/06188; WO 92/06200; WO 96/10640; Barnes, W. M.,
Gene 112:29-35 (1992); Lawyer, F. C., et al., PCR Meth. Appl.
2:275-287 (1993); Flaman, J.-M, et al., Nucl. Acids Res.
22(15):3259-3260 (1994)). For amplification of long nucleic acid
molecules (e.g., nucleic acid molecules longer than about 3-5 Kb in
length), at least two DNA polymerases (one substantially lacking 3'
exonuclease activity and the other having 3' exonuclease activity)
are typically used. See U.S. Pat. No. 5,436,149; and U.S. Pat. No.
5,512,462; Barnes, W. M., Gene 112:29-35 (1992), the disclosures of
which are incorporated herein in their entireties. Examples of DNA
polymerases substantially lacking in 3' exonuclease activity
include, but are not limited to, Taq, Tne(exo-), Tma(exo-), Pfu
(exo-), Pwo(exo-) and Tth DNA polymerases, and mutants, variants
and derivatives thereof.
[0112] DNA polymerases for use in the present invention may be
obtained commercially, for example, from Life Technologies, Inc.
(Rockville, Md.), Pharmacia (Piscataway, N.J.), Sigma (St. Louis,
Mo.) and Boehringer Mannheim. Preferred DNA polymerases for use in
the present invention include Tsp DNA polymerase from Life
Technologies, Inc.
[0113] Enzymes for use in the compositions, methods and kits of the
invention include any enzyme having reverse transcriptase activity.
Such enzymes include, but are not limited to, retroviral reverse
transcriptase, retrotransposon reverse transcriptase, hepatitis B
reverse transcriptase, cauliflower mosaic virus reverse
transcriptase, bacterial reverse transcriptase, Tth DNA polymerase,
Taq DNA polymerase (Saiki, R. K., et al., Science 239:487-491
(1988); U.S. Pat. Nos. 4,889,818 and 4,965,188), Tne DNA polymerase
(WO 96/10640), Tma a DNA polymerase (U.S. Pat. No. 5,374,553) and
mutants, fragments, variants or derivatives thereof (see, e.g.,
commonly owned, co-pending U.S. patent application Ser. Nos.
08/706,702 and 08/706,706, both filed Sep. 9, 1996, which are
incorporated by reference herein in their entireties). As will be
understood by one of ordinary skill in the art, modified reverse
transcriptases and DNA polymerase having RT activity may be
obtained by recombinant or genetic engineering techniques that are
well-known in the art. Mutant reverse transcriptases or polymerases
can, for example, be obtained by mutating the gene or genes
encoding the reverse transcriptase or polymerase of interest by
site-directed or random mutagenesis. Such mutations may include
point mutations, deletion mutations and insertional mutations.
Preferably, one or more point mutations (e.g., substitution of one
or more amino acids with one or more different amino acids) are
used to construct mutant reverse transcriptases or polymerases for
use in the invention. Fragments of reverse transcriptases or
polymerases may also be obtained by deletion mutation by
recombinant techniques that are well-known in the art, or by
enzymatic digestion of the reverse transcriptase(s) or
polymerase(s) of interest using any of a number of well-known
proteolytic enzymes.
[0114] Preferred enzymes for use in the invention include those
that are reduced or substantially reduced in RNase H activity. Such
enzymes that are reduced or substantially reduced in RNase H
activity may be obtained by mutating the RNase H domain within the
reverse transcriptase of interest, preferably by one or more point
mutations, one or more deletion mutations, and/or one or more
insertion mutations as described above. By an enzyme "substantially
reduced in RNase H activity" is meant that the enzyme has less than
about 30%, less than about 25%, less than about 20%, more
preferably less than about 15%, less than about 10%, less than
about 7.5%, or less than about 5%, and most preferably less than
about 5% or less than about 2%, of the RNase H activity of the
corresponding wildtype or RNase H.sup.+ enzyme such as wildtype
Moloney Murine Leukemia Virus (M-MLV), Avian Myeloblastosis Virus
(AMV) or Rous Sarcoma Virus (RSV) reverse transcriptases. The RNase
H activity of any enzyme may be determined by a variety of assays,
such as those described, for example, in U.S. Pat. No. 5,244,797,
in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988), in
Gerard, G. F., et al., FOCUS 14(5):91 (1992), and in U.S. Pat. No.
5,668,005, the disclosures of all of which are fully incorporated
herein by reference.
[0115] Polypeptides having reverse transcriptase activity for use
in the invention may be obtained commercially, for example from
Life Technologies, Inc. (Rockville, Md.), Pharmacia (Piscataway,
N.J.), Sigma (Saint Louis, Mo.) or Boehringer Mannheim Biochemicals
(Indianapolis, Ind.). Alternatively, polypeptides having reverse
transcriptase activity may be isolated from their natural viral or
bacterial sources according to standard procedures for isolating
and purifying natural proteins that are well-known to one of
ordinary skill in the art (see, e.g., Houts. G. E., et al., J.
Virol. 29:517 (1979)). In addition, the polypeptides having reverse
transcriptase activity may be prepared by recombinant DNA
techniques that are familiar to one of ordinary skill in the art
(see, e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265
(1988); Soltis, D. A., and Skalka, A. M., Proc. Natl. Acad. Sci.
USA 85:3372-3376 (1988)).
[0116] Preferred polypeptides having reverse transcriptase activity
for use in the invention include M-MLV reverse transcriptase, RSV
reverse transcriptase, AMV reverse transcriptase, Rous Associated
Virus (RAV) reverse transcriptase, Myeloblastosis Associated Virus
(MAV) reverse transcriptase and Human Immunodeficiency Virus (HIV)
reverse transcriptase, and others described in WO 98/47921 and
derivatives, variants, fragments or mutants thereof, and
combinations thereof. In a further preferred embodiment, the
reverse transcriptases are reduced or substantially reduced in
RNase activity, and are most preferably selected from the group
consisting of M-MLV H.sup.- reverse transcriptase, RSV H.sup.- if
reverse transcriptase, AMV H.sup.- reverse transcriptase, RAV
H.sup.- reverse transcriptase, MAV H.sup.- reverse transcriptase
and HIV H.sup.- reverse transcriptase, and derivatives, variants,
fragments or mutants thereof, and combinations thereof. Reverse
transcriptases of particular interest include AMV RT and M-MLV RT,
and more preferably AMV RT and M-MLV RT having reduced or
substantially reduced RNase H.sup.- activity (preferably AMV RT
.alpha.H.sup.-/BH.sup.- and M-MLV RT H.sup.-). The most preferred
reverse transcriptases for use in the invention include
SuperScript.TM., SuperScript.TM. II, ThermoScript.TM. and
ThermoScript.TM. II available from Life Technologies, Inc. See
generally, WO 98/47921, U.S. Pat. Nos. 5,244,797 and 5,668,005, the
entire contents of each of which are herein incorporated by
reference.
[0117] Hairpin. As used herein, the term "hairpin" is used to
indicate the structure of an oligonucleotide in which one or more
portions of the oligonucleotide form base pairs with one or more
other portions of the oligonucleotide. When the two portions are
base paired to form a double stranded portion of the
oligonucleotide, the double stranded portion may be referred to as
a stem. Thus, depending on the number of complementary portions
used, a number of stems (preferably 1-10) may be formed.
Additionally, formation of the one or more stems preferably allows
formation of one or more loop structures in the hairpin molecule.
In one aspect, any one or more of the loop structures may be cut or
nicked at one or more sites within the loop or loops but preferably
at least one loop is not so cut or nicked. The sequence of the
oligonucleotide may be selected so as to vary the number of
nucleotides which base pair to form the stem from about 3
nucleotides to about 100 or more nucleotides, from about 3
nucleotides to about 50 nucleotides, from about 3 nucleotides to
about 25 nucleotides, and from about 3 to about 10 nucleotides. In
addition, the sequence of the oligonucleotide may be varied so as
to vary the number of nucleotides which do not form base pairs from
0 nucleotides to about 100 or more nucleotides, from 0 nucleotides
to about 50 nucleotides, from 0 nucleotides to about 25 nucleotides
or from 0 to about 10 nucleotides. The two portions of the
oligonucleotide which base pair may be located anywhere or at any
number of locations in the sequence of the oligonucleotide. In some
embodiments, one base-pairing-portion of the oligonucleotide may
include the 3'-terminal of the oligonucleotide. In some
embodiments, one base-pairing-portion may include the 5'-terminal
of the oligonucleotide. In some embodiments, one
base-pairing-portion of the oligonucleotide may include the
3'-terminal while the other base-pairing-portion may include the
5'-terminal and, when base paired, the stem of the oligonucleotide
is blunt ended. In other embodiments, the location of the base
pairing portions of the oligonucleotide may be selected so as to
form a 3'-overhang, a 5'-overhang and/or may be selected so that
neither the 3'-nor the 5'-most nucleotides are involved in base
pairing.
[0118] Hybridization. As used herein, the terms "hybridization" and
"hybridizing" refer to the pairing of two complementary
single-stranded nucleic acid molecules (RNA and/or DNA) to give a
double-stranded molecule. As used herein, two nucleic acid
molecules may be hybridized, although the base pairing is not
completely complementary. Accordingly, mismatched bases do not
prevent hybridization of two nucleic acid molecules provided that
appropriate conditions, well known in the art, are used.
[0119] Incorporating. The term "incorporating" as used herein means
becoming a part of a DNA or RNA molecule or primer.
[0120] Nucleotide. As used herein "nucleotide" refers to a
base-sugar-phosphate combination. Nucleotides are monomeric units
of a nucleic acid sequence (DNA and RNA). The term nucleotide
includes mono-, di- and triphosphate forms of deoxyribonucleosides
and ribonucleosides and their derivatives. The term nucleotide
particularly includes deoxyribonucleoside triphosphates such as
dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such
derivatives include, for example, [.alpha.S]dATP, 7-deaza-dGTP and
7-deaza-dATP. The term nucleotide as used herein also refers to
dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
Illustrated examples of dideoxyribonucleoside triphosphates
include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and
ddTTP. According to the present invention, a "nucleotide" may be
unlabeled or detectably labeled by well known techniques.
Detectable labels include, for example, radioactive isotopes,
fluorescent labels, chemiluminescent labels, bioluminescent labels
and enzyme labels.
[0121] Oligonucleotide. As used herein, "oligonucleotide" refers to
a synthetic or biologically produced molecule comprising a
covalently linked sequence of nucleotides which may be joined by a
phosphodiester bond between the 3' position of the pentose of one
nucleotide and the 5' position of the pentose of the adjacent
nucleotide. Oligonucleotide as used herein is seen to include
natural nucleic acid molecules (i.e., DNA and RNA) as well as
non-natural or derivative molecules such as peptide nucleic acids,
phophothioate containing nucleic acids, phosphonate containing
nucleic acids and the like. In addition, oligonucleotides of the
present invention may contain modified or non-naturally occurring
sugar residues (i.e., arabainose) and/or modified base residues.
Oligonucleotide is seen to encompass derivative molecules such as
nucleic acid molecules comprising various natural nucleotides,
derivative nucleotides, modified nucleotides or combinations
thereof. Thus any oligonucleotide or other molecule useful in the
methods of the invention are contemplated by this definition.
Oligonucleotides of the present invention may also comprise
blocking groups which prevent the interaction of the molecule with
particular proteins, enzymes or substrates.
[0122] Primer. As used herein, "primer" refers to a synthetic or
biologically produced single-stranded oligonucleotide that is
extended by covalent bonding of nucleotide monomers during
amplification or polymerization of a nucleic acid molecule. Nucleic
acid amplification often is based on nucleic acid synthesis by a
nucleic acid polymerase or reverse transcriptase. Many such
polymerases or reverse transcriptases require the presence of a
primer that can be extended to initiate such nucleic acid
synthesis. A primer is typically 11 bases or longer; most
preferably, a primer is 17 bases or longer, although shorter or
longer primers may be used depending on the need. As will be
appreciated by those skilled in the art, the oligonucleotides of
the invention may be used as one or more primers in various
extension, synthesis or amplification reactions.
[0123] Probe. As used herein, "probe" refers to synthetic or
biologically produced nucleic acids (DNA or RNA) which, by design
or selection, contain specific nucleotide sequences that allow them
to hybridize, under defined stringencies, specifically (i.e.,
preferentially) to target nucleic acid sequences. As will be
appreciated by those skilled in the art, the oligonucleotides of
the present invention may be used as one or more probes and
preferably may be used as probes for the detection or
quantification of nucleic acid molecules.
[0124] Substantially less extendable. As used herein,
"substantially less extendable" is used to characterize an
oligonucleotide that is inefficiently extended or not extended in
an extension and/or amplification reaction when the 3'-most
nucleotide of the oligonucleotide is not complementary to the
corresponding base of a target/template nucleic acid. Preferably,
an oligonucleotide is substantially less extendable as a result of
the presence of a specificity enhancing group on the
oligonucleotide. In this event, an oligonucleotide is substantially
less extendable when the oligonucleotide is not extended or is
extended by a lesser amount and/or at a slower rate than an
oligonucleotide lacking the specificity enhancing group but having
an otherwise identical structure. Those skilled in the art can
readily determine if an oligonucleotide is substantially less
extendable by conducting an extension reaction using an
oligonucleotide containing a specificity enhancing group and
comparing the extension to the extension of an oligonucleotide of
the same structure but lacking the specificity enhancing group.
Under identical extension conditions, (e.g., melting temperature
and time, annealing temperature and time, extension temperature and
time, reactant concentrations and the like), a substantially less
extendable oligonucleotide will produce less extension product when
the 3'-most nucleotide of the oligonucleotide is not complementary
to the corresponding nucleotide on a target/template nucleic acid
than will be produced by an oligonucleotide lacking a specificity
enhancing group but having an otherwise identical structure.
Alternatively, one skilled in the art can determine if an
oligonucleotide is substantially less extendable by conducting
allele specific PCR with a first set of oligonucleotides at least
one of which comprises one or more specificity enhancing groups and
with a second set of oligonucleotides lacking specificity enhancing
groups but otherwise of identical structure to those of the first
set. Then a determination is separately made for each set of
primers of the difference in the amount of product made and/or the
rate at which the product is made with the oligonucleotide having
the 3'-nucleotide complementary to the corresponding nucleotide on
a target/template nucleic acid to the amount of product made and/or
the rate at which the product is made with an oligonucleotide
having the 3'-nucleotide not complementary to the corresponding
nucleotide on a target/template nucleic acid. Substantially less
extendable oligonucleotides will produce a larger difference in
amount of product made and/or rate at which product is made between
3'-complementary and 3'-not-complementary oligonucleotides.
Preferably the difference in the amount of product made and/or rate
at which product is made using oligonucleotides containing
specificity enhancing groups will be between from about 1.1 fold to
about 1000 fold larger than the difference obtained using primers
lacking specificity enhancing groups, or from about 1.1 fold to
about 500 fold larger, or from about 1.1 fold to about 250 fold
larger, or from about 1.1 fold to about 100 fold larger, or from
about 1.1 fold to about 50 fold larger, or from about 1.1 to about
25 fold larger, or from about 1.1 to about 10 fold larger, or from
about 1.1 fold to about 5 fold or from about 1.1 fold to about 2
fold larger. The amount of product can be determined using any
methodology known to those of skill in the art, for example, by
running the product on an agarose gel and staining with ethidium
bromide and comparing to known amounts of similarly treated nucleic
acid standards. The amount of product may be determined at any
convenient time point in the allele specific PCR. One convenient
way to compare the rate of formation of product is to compare the
number of cycles required to form a specified amount of product in
a PCR. A determination is separately made for each set of primers
of the difference between the number of cycles required to make a
given amount of product with the oligonucleotide having the
3'-nucleotide complementary to the corresponding nucleotide on a
target/template nucleic acid and the number of cycles required to
make the same amount of product with an oligonucleotide having the
3'-nucleotide not complementary to the corresponding nucleotide on
a target/template nucleic acid. Substantially less extendable
oligonucleotides will produce a larger difference in the number of
cycles required to produce a specified amount of product between
3'-complementary and 3'-not-complementary oligonucleotides. The
amount of product made can be determined using any means known to
those skilled in the art, for example, by determining the
fluorescence intensity of a labeled product using a thermocycler
adapted to perform real time fluorescence detection. Preferably the
difference between the number of cycles required to make a
specified amount of product using oligonucleotides containing
specificity enhancing groups will be between from about 1.05 fold
to about 100 fold larger than the difference obtained using primers
lacking specificity enhancing groups, or from about 1.05 fold to
about 50 fold larger, or from about 1.05 fold to about 25 fold
larger, or from about 1.05 fold to about 10 fold larger, or from
about 1.05 fold to about 5 fold larger, or from about 1.05 to about
2.5 fold larger, or from about 1.05 to about 1.5 fold larger, or
from about 1.05 fold to about 1.2 fold larger.
[0125] Support. As used herein a "support" may be any material or
matrix suitable for attaching the oligonucleotides of the present
invention or target/template nucleic acid sequences. Such
oligonucleotides and/or sequences may be added or bound (covalently
or non-covalently) to the supports of the invention by any
technique or any combination of techniques well known in the art.
Supports of the invention may comprise nitrocellulose,
diazocellulose, glass, polystyrene (including microtitre plates),
polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose,
agar, starch and nylon. Supports of the invention may be in any
form or configuration including beads, filters, membranes, sheets,
frits, plugs, columns and the like. Solid supports may also include
multi-well tubes (such as microtitre plates) such as 12-well
plates, 24-well plates, 48-well plates, 96-well plates, and
384-well plates. Preferred beads are made of glass, latex or a
magnetic material (magnetic, paramagnetic or superparamagnetic
beads).
[0126] In a preferred aspect, methods of the invention may be used
in conjunction with arrays of nucleic acid molecules (RNA or DNA).
Arrays of nucleic acid template/target or arrays of
oligonucleotides of the invention are both contemplated in the
methods of the invention. Such arrays may be formed on microplates,
glass slides or standard blotting membranes and may be referred to
as microarrays or gene-chips depending on the format and design of
the array. Uses for such arrays include gene discovery, gene
expression profiling and genotyping (SNP analysis,
pharmacogenomics, toxicogenetics).
[0127] Synthesis and use of nucleic acid arrays and generally
attachment of nucleic acids to supports have been described (see
for example, U.S. Pat. No. 5,436,327, U.S. Pat. No. 5,800,992, U.S.
Pat. No. 5,445,934, U.S. Pat. No. 5,763,170, U.S. Pat. No.
5,599,695 and U.S. Pat. No. 5,837,832). An automated process for
attaching various reagents to positionally defined sites on a
substrate is provided in Pirrung et al. U.S. Pat. No. 5,143,854 and
Barrett et al. U.S. Pat. No. 5,252,743.
[0128] Essentially, any conceivable support may be employed in the
invention. The support may be biological, nonbiological, organic,
inorganic, or a combination of any of these, existing as particles,
strands, precipitates, gels, sheets, tubing, spheres, containers,
capillaries, pads, slices, films, plates, slides, etc. The support
may have any convenient shape, such as a disc, square, sphere,
circle, etc. The support is preferably flat but may take on a
variety of alternative surface configurations. For example, the
support may contain raised or depressed regions on which one or
more methods of the invention may take place. The support and its
surface preferably form a rigid support on which to carry out the
reactions described herein. The support and its surface is also
chosen to provide appropriate light-absorbing characteristics. For
instance, the support may be a polymerized Langmuir Blodgett film,
functionalized glass, Si, Ge, GaAs, GaP, SiO.sub.2, SIN.sub.4,
modified silicon, or any one of a wide variety of gels or polymers
such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,
polystyrene, polycarbonate, or combinations thereof. Other support
materials will be readily apparent to those of skill in the art
upon review of this disclosure. In a preferred embodiment the
support is flat glass or single-crystal silicon.
[0129] Target molecule. As used herein, "target molecule" refers to
a nucleic acid molecule to which a particular primer or probe is
capable of preferentially hybridizing.
[0130] Target sequence. As used herein, "target sequence" refers to
a nucleic acid sequence within the target molecules to which a
particular primer or probe is capable of preferentially
hybridizing.
[0131] Template. The term "template" as used herein refers to a
double-stranded or single-stranded molecule which is to be
amplified, synthesized or sequenced. In the case of a
double-stranded DNA molecule, denaturation of its strands to form a
first and a second strand is preferably performed to amplify,
sequence or synthesize these molecules. A primer, complementary to
a portion of a template is hybridized under appropriate conditions
and the polymerase (DNA polymerase or reverse transcriptase) may
then synthesize a nucleic acid molecule complementary to said
template or a portion thereof. The newly synthesized molecule,
according to the invention, may be equal or shorter in length than
the original template. Mismatch incorporation during the synthesis
or extension of the newly synthesized molecule may result in one or
a number of mismatched base pairs. Thus, the synthesized molecule
need not be exactly complementary to the template. The template can
be an RNA molecule, a DNA molecule or an RNA/DNA hybrid molecule. A
newly synthesized molecule may serve as a template for subsequent
nucleic acid synthesis or amplification.
[0132] Thermostable. As used herein "thermostable" refers to a
polymerase (RNA, DNA or RT) which is resistant to inactivation by
heat. DNA polymerases synthesize the formation of a DNA molecule
complementary to a single-stranded DNA template by extending a
primer in the 5'-to-3' direction. This activity for mesophilic DNA
polymerases may be inactivated by heat treatment. For example, T5
DNA polymerase activity is totally inactivated by exposing the
enzyme to a temperature of 90.degree. C. for 30 seconds. As used
herein, a thermostable DNA polymerase activity is more resistant to
heat inactivation than a mesophilic DNA polymerase. However, a
thermostable DNA polymerase does not mean to refer to an enzyme
which is totally resistant to heat inactivation and thus heat
treatment may reduce the DNA polymerase activity to some extent. A
thermostable DNA polymerase typically will also have a higher
optimum temperature than mesophilic DNA polymerases.
[0133] Other terms used in the fields of recombinant DNA technology
and molecular and cell biology as used herein will be generally
understood by one of ordinary skill in the applicable arts.
[0134] The present invention provides oligonucleotides, which may
be labeled internally, and/or, at or near the 3' termini and/or 5'
termini or may be unlabeled. In another aspect, the
oligonucleotides of the present invention may be provided with a
specificity enhancing group. Such a group may be located internally
and/or at or near the 3'- and/or the 5'-terminal of the
oligonucleotide. In another aspect, the oligonucleotides of the
present invention may be in the form of a hairpin. In some
preferred embodiments, the oligonucleotides may be provided with
more than one of these characteristics, i.e., they may comprise a
label and/or a specificity enhancing group and/or may be in the
form of a hairpin.
[0135] When labeled, oligonucleotides of the invention may contain
one or multiple labels (which may be the same or different). The
oligonucleotides of the invention may be used as primers and/or
probes. In a preferred aspect, the oligonucleotides are labeled and
the label is any moiety which undergoes a detectable change in any
observable property upon hybridization and/or extension. In a
preferred embodiments, the label is a fluorescent moiety and the
label undergoes a detectable change in one or more fluorescent
properties. Such properties are seen to include, but are not
limited to, fluorescent intensity, fluorescent polarization,
fluorescent lifetime and quantum yield of fluorescence. The
oligonucleotides for use in the invention can be any suitable size,
and are preferably in the range of 10-100 or 10-80 nucleotides,
more preferably 11-40 nucleotides and most preferably in the range
of 17-25 nucleotides although oligonucleotides may be longer or
shorter depending upon the need.
[0136] The oligonucleotides of the invention can be DNA or RNA or
chimeric mixtures or derivatives or modified versions thereof. In
addition to being labeled with a detectable moiety, the
oligonucleotide can be modified at the base moiety, sugar moiety,
or phosphate backbone, and may include other appending groups or
labels.
[0137] For example, the oligonucleotides of the invention may
comprise at least one modified or more base moieties which are
selected from the group including but not limited to
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylamino-methyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.sup.6-isopentenyladenine,
1-methylguanine, 1-methyl-linosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N.sup.6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-me thoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxy-methyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0138] In another embodiment, the oligonucleotides of the invention
comprises at least one modified sugar moiety selected from the
group including but not limited to arabinose, 2-fluoroarabinose,
xylulose, and hexose.
[0139] In yet another embodiment, the oligonucleotides of the
invention comprises at least one modified phosphate backbone
selected from the group consisting of a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0140] The oligonucleotides of the invention have use in nucleic
acid amplification or synthesis reactions (e.g., as primers) to
detect or measure a nucleic acid product of the amplification or
synthesis reaction, thereby detecting or measuring a target nucleic
acid in a sample that is complementary to all or a portion of a
primer sequence. The oligonucleotides of the invention may be used
in any amplification reactions including PCR, 5-RACE, Anchor PCR,
"one-sided PCR," LCR, NASBA, SDA, RT-PCR and other amplification
systems known in the art.
[0141] Thus, the invention generally relates to methods of
synthesizing or amplifying one or more nucleic acid molecules
comprising: [0142] (a) mixing one or more templates or target
nucleic acid molecules with one or more oligonucleotides of the
invention; and [0143] (b) incubating said mixture under conditions
sufficient to synthesize or amplify one or more nucleic acid
molecules complementary to all or a portion of said templates or
target molecules. Preferably, the synthesized or amplified nucleic
acid molecules comprise one or more oligonucleotides of the
invention or portions thereof. In one aspect, the oligonucleotides
of the invention are incorporated at or near one or both termini of
the synthesized or amplified nucleic acid molecules produced by the
methods of the invention. The invention also relates to one or more
nucleic acid molecules produced by such amplification or synthesis
reactions.
[0144] In another aspect, the invention relates to methods of
synthesizing one or more nucleic acid molecules, comprising [0145]
(a) mixing one or more nucleic acid templates (which may be DNA
molecules such as a cDNA molecules, or RNA molecules such as mRNA
molecules, or populations of such molecules) with one or more
primers of the invention and one or more polymerases; and [0146]
(b) incubating the mixture under conditions sufficient to
synthesize one or more first nucleic acid molecules complementary
to all or a portion of the templates. Such incubation conditions
may involve the use of one or more nucleotides and one or more
nucleic acid synthesis buffers. Such methods of the invention may
optionally comprise one or more additional steps, such as
incubating the synthesized first nucleic acid molecules under
conditions sufficient to make one or more second nucleic acid
molecules complementary to all or a portion of the first nucleic
acid molecules. Such additional steps may also be accomplished in
the presence of one or more primers of the invention and one or
more polymerases as described herein. The invention also relates to
nucleic acid molecules synthesized by these methods.
[0147] The invention also relates to methods for sequencing nucleic
acid molecules comprising [0148] (a) mixing a nucleic acid molecule
to be sequenced with one or more primers of the invention, one or
more nucleotides and one or more terminating agents to form a
mixture; [0149] (b) incubating the mixture under conditions
sufficient to synthesize the population of molecules complementary
to all or a portion of the molecule to be sequence; and [0150] (c)
separating the population to determining the nucleotide sequence of
all or a portion of the molecule to be sequenced.
[0151] The invention more specifically relates to a method of
sequencing a nucleic acid molecule, comprising: [0152] (a) mixing
one or more of the oligonucleotides of the invention, one or more
nucleotides, and one or more terminating agents; [0153] (b)
hybridizing said oligonucleotides to a first nucleic acid molecule;
[0154] (c) incubating the mixture of step (b) under conditions
sufficient to synthesize a random population of nucleic acid
molecules complementary to said first nucleic acid molecule,
wherein said synthesized molecule are shorter in length than said
first molecule and wherein said synthesized molecules comprise a
terminator nucleotide at their 3' termini; and [0155] separating
said synthesized molecules by size so that at least a part of the
nucleotide sequence of said first nucleic acid molecule can be
determined. Such terminator nucleotides include ddTTP, ddATP,
ddGTP, ddITP or ddCTP. Such incubation conditions may include
incubation in the presence of one or more polymerases and/or
buffering salts.
[0156] In a related aspect, the oligonucleotides of the invention
are useful in detecting the presence or absence of or quantifying
the amount of nucleic acid molecules in a sample without the need
for performing amplification or synthesis reactions. In accordance
with the invention, an oligonucleotide may be provided with one or
more labels which undergo a detectable change in at least one
observable property when the oligonucleotide comprising the label
is converted to a double stranded molecule (e.g., by hybridizing
the oligonucleotide to a target molecule). Thus, a change in an
observable property indicates the presence of the target molecule
in the sample when compared to a control sample not containing the
nucleic acid molecule of interest. Quantification of the nucleic
acid target molecule in the sample may also be determined by
comparing change in the observable property in an unknown sample to
the changes in the observable property in samples containing known
amounts of the nucleic acid target molecule of interest. Any
samples thought to contain the nucleic acid molecule of interest
may be used including, but not limited to, biological samples such
as blood, urine, tissue, cells, feces, serum, plasma, or any other
samples derived from animals (including humans), plants, bacteria,
viruses and the like. Environmental samples such as soil samples,
water samples, air samples and the like may also be used in
accordance with the invention.
[0157] The oligonucleotides of the invention can be used in methods
of diagnosis, wherein the oligonucleotide is complementary to a
sequence (e.g., genomic or cDNA) of an infectious disease agent or
is capable of initiating synthesis or amplification of a sequence
of an infectious disease agent, e.g. of human disease including but
not limited to viruses (e.g, HIV, HPV etc), bacteria, parasites,
and fungi, thereby diagnosing the presence of the infectious agent
in a sample from a patient. The type of target nucleic acid can be
genomic, cDNA, mRNA, synthetic, or the source may be human, animal,
or bacterial. In another embodiment that can be used in the
diagnosis or prognosis of a disease or disorder, the target
sequence is a wild type human genomic or RNA or cDNA sequence,
mutation of which is implicated in the presence of a human disease
or disorder, or alternatively, can be the mutated sequence. In such
an embodiment, the hybridization, amplification or synthesis
reaction of the invention can be repeated for the same sample with
different sets of oligonucleotides of the invention (for example,
with differently labeled oligonucleotide) which selectively
identify the wild type sequence or the mutated version. By way of
example, the mutation can be an insertion, substitution, and/or
deletion of one or more nucleotides, or a translocation.
[0158] In a specific embodiment, the invention provides a method
for detecting or measuring a product of a nucleic acid
amplification or synthesis reaction comprising (a) contacting a
sample comprising one or more target nucleic acid molecules with
one or more primers (such primers may comprise one or multiple
labels, which may be the same or different and may be labeled
internally, and/or, at or near the 3' and/or 5' end), said primers
being adapted for use in said amplification or synthesis reaction
such that said primers are incorporated into an amplified or
synthesized product of said amplification or synthesis reaction
when a target sequence or nucleic acid molecule is present in the
sample; (b) conducting the amplification or synthesis reaction; and
(c) detecting or measuring one or more synthesis or amplification
product molecules (preferably by detecting a change in one or more
observable properties of one or more labels).
[0159] In another specific embodiment, the invention provides for a
method of detecting or measuring the presence or absence or the
amount of a target nucleic acid molecule within a sample comprising
(a) contacting a sample comprising one or more target nucleic acid
molecules with one or more oligonucleotides of the invention (such
oligonucleotides may comprise one or multiple labels, which may be
the same or different and may be labeled internally and/or at or
near the 3' and/or 5' end); (b) incubating said mixture under
conditions sufficient to allow said oligonucleotides to interact
with said target molecules sufficient to form double stranded
molecules (preferably through hybridization); and (c) detecting one
or more of said target nucleic acid molecules (preferably by
detecting a change in one or more observable properties of one or
more labels).
[0160] The present invention provides a method for detecting a
target nucleic acid sequence, comprising the steps of contacting a
sample containing a mixture of nucleic acids with at least one
oligonucleotide of the present invention, the oligonucleotide
capable of hybridizing a target nucleic acid sequence and comprises
at least one detectable moiety, wherein the detectable moiety
undergoes a change in one or more observable properties upon
hybridization to the target nucleic acid sequence and observing the
observable property, wherein a change in the observable property
indicates the presence of the target nucleic acid sequence. In some
embodiments, the target nucleic acid sequence is not separated from
the mixture. In some embodiments, the observable property is
fluorescence. In some embodiments, the change is an increase in
fluorescence. In some embodiments, the change is a decrease in
fluorescence. In some embodiments, the oligonucleotide comprises a
specificity enhancing group. In some embodiments, the
oligonucleotide is in the form of a hairpin.
[0161] The present invention provides a method for quantifying a
target nucleic acid molecule, comprising the steps contacting a
sample containing a mixture of nucleic acids comprising the target
nucleic acid molecule with at least one oligonucleotide of the
present invention, the oligonucleotide capable 15 of hybridizing to
the target nucleic acid molecule and comprises at least one
detectable moiety, wherein the detectable moiety undergoes a change
in one or more observable properties upon hybridization to the
target nucleic acid sequence and observing the observable property,
wherein a change in the observable property is proportional to the
amount of the target nucleic acid molecule in the sample.
[0162] In a further aspect, the invention relates to the use of one
or more treatments to lower or decrease the energy emitted by the
labels of the oligonucleotides of the invention. Such treatments
may be used in accordance with the invention to lower the
background in the hybridization, synthesis or amplification methods
of the invention. In one aspect, single stranded nucleic acid
binding protein (E. coli, T4 bacteriophage or Archaea (see Kelly,
et al. Proceedings of the National Academy of Sciences, USA
95:14634-14639 (1998), Chedin, et al., TIBS 23:273-277 (1998), U.S.
Pat. Nos. 5,449,603, 5,605,824, 5,646,019, and 5,773,257) may be
used to interact with single stranded labeled oligonucleotides of
the invention to reduce or quench energy emitted or other
detectable properties from the labels. Such single stranded binding
proteins may be native or modified. During the detection or
quantitation process (hybridization, synthesis or amplification
reactions) double stranded nucleic acid molecules formed do not
substantially interact with single stranded binding protein or
interact minimally with such double stranded molecules.
Accordingly, in the unreacted state (single stranded form of the
oligonucleotides of the invention), energy emitted or other
detectable properties (e.g., fluorescence) is reduced or quenched
while in the reactive form (double stranded molecules) energy
emitted or other detectable properties is enhanced. In another
aspect, blocking oligonucleotides which contain quencher molecules
may be used to competitively bind the labeled oligonucleotides in
the invention in the unreacted stated thereby reducing energy
emitted or other detectable properties of the labeled
oligonucleotide. In another aspect, one or more additional
fluorescent moieties may be incorporated into the blocking molecule
such that the fluorescent moiety on the oligonucleotide of the
invention is in proximity to the one or more additional fluorescent
moieties when the oligonucleotide of the invention is in the
unreacted state. The presence of an additional fluorescent molecule
can reduce the background fluorescence level even though there is
little or no overlap between the emission spectrum of the
fluorescent moiety on the oligonucleotide of the invention and the
absorption spectrum of the one or more additional fluorescent
moieties on the blocking oligonucleotide. When the oligonucleotide
of the invention has the capability of forming a hairpin structure,
those skilled in the art will appreciate the one or more additional
fluorescent moieties can be brought into proximity with the label
on the oligonucleotide of the invention by attaching the one or
more additional fluorescent moieties to nucleotides in one strand
of the stem structure of the hairpin while attaching one or more
labels to nucleotides in the other strand. During detection or
quantitation, target nucleic acid molecules interact with labeled
oligonucleotides of the invention thereby enhancing energy emitted
or other detectable properties by the labels. Such interaction may
separate the blocking oligonucleotide (e.g., quencher/additional
fluorescent moiety-containing molecule) from the label containing
oligonucleotide of the invention.
[0163] In another aspect of the present invention, the sequence of
the oligonucleotide and/or a blocking oligonucleotide may be
selected so as to reduce the background fluorescence of the
oligonucleotides of the invention. It has been unexpectedly found
that the base sequence in the vicinity of the label can have a
dramatic effect on the background fluorescence level. The
background fluorescence of a single stranded oligonucleotide of the
present invention can be decreased about 5 fold if the sequence of
the oligonucleotide is selected so as to form a blunt-end double
stranded structure with one or more fluorophores located on one or
more base close to the 3'-end and G-C or C-G base pair being the
last base pair of the double stranded structure. In some preferred
embodiments, the double stranded structure may be a stem of a
hairpin structure. In some preferred embodiments, the 3'-end of the
oligonucleotides of the invention may be provide with one of the
following sequences: 5'- . . . T(Fluo)C-3', 5'- . . . T(Fluo)G-3',
5'- . . . T(Fluo)AG-3', 5'- . . . T(Fluo)AC-3', 5'- . . .
T(Fluo)TC-3', 5'- . . . T(Fluo)TG-3' where the attachment of a
fluorophore is indicated by (Fluo) and the 3'-sequence is as shown
while the blocking oligonucleotide (or 5'-end of a hairpin
oligonucleotide) is provided with the complementary sequence
(preferably at the 5' end of the blocking oligonucleotide/hairpin
molecule). To achieve a quenching effect the labeled base should be
within 10 nucleotides distance from the 3'-end, preferably within 6
nucleotides and most preferably within 1-4 nucleotides. A specific
example of oligonucleotides of this type is provided by Oligo 10
(SEQ ID NO:22) in Table 2. In a related embodiment, when using an
oligonucleotide that does not have G or C for its 3'-most
nucleotide and hence cannot form a G-C base pair at the 3'-end, the
addition of a 5'-overhanging G residue to the oligonucleotide can
reduce the background fluorescence. Also, the presented mode of
quenching can be combined with another mechanism of quenching like
fluorescence resonance energy transfer or static quenching. In some
embodiments of the present invention, combinations of quenching
techniques may be employed to reduce the background fluorescence.
For example, an oligonucleotide of the present invention may have a
detectable moiety located near the 3' end of the oligonucleotide
while the sequence of the oligonucleotide may be selected so as to
have a G-C base pair at a blunt end of a hairpin structure and one
or more additional fluorescent moieties may be attached to
nucleotides at or near the 5'-end of the oligonucleotide. A similar
structure could be employed utilizing a blocking oligonucleotide
instead of a hairpin.
[0164] Other means for quenching or reducing nonreacted labeled
oligonucleotidcs may be used or any combination of such treatments
may be used in accordance with the invention.
[0165] The present invention provides a composition comprising one
or more oligonucleotides of the invention and one or more target or
template nucleic acid molecules, wherein at least a portion of the
oligonucleotide is capable of hybridizing to at least a portion of
the target or template nucleic acid molecule (preferably the
oligonucleotide comprises one or more detectable moieties that
undergo a change in one or more observable property upon
hybridization to the target nucleic acid molecule). In some
embodiments, the detectable moiety is a fluorescent moiety and the
fluorescent moiety undergoes a change in fluorescence upon
hybridizing to the target nucleic acid molecule. In some
embodiments, the oligonucleotide is a hairpin when not hybridized
to the target nucleic acid molecule.
[0166] In some preferred embodiments, the present invention
provides a composition comprising at least one nucleic acid
molecule and at least one oligonucleotide of the invention, wherein
at least a portion of said oligonucleotide is capable of
hybridizing with at least a portion of said nucleic acid molecule
and wherein said oligonucleotide comprises one or more specificity
enhancing groups. In some embodiments, one or more of the
specificity enhancing groups may be a fluorescent moiety. A
specificity enhancing group may be attached at any position of the
oligonucleotide that results in the oligonucleotide being
substantially less extendable when the 3'most nucleotide of the
oligonucleotide is not complementary to the corresponding
nucleotide of a target/template nucleic acid. In some embodiments,
at least one of the one or more groups is attached to a nucleotide
at or near the 3'-nucleotide. In some embodiments, at least one of
the one or more groups is attached to one, of the ten 3'-most
nucleotides. In other words, in embodiments of this type, at least
one of the one or more specificity enhancing groups may be attached
to the 3'-most nucleotide or any of the next nine contiguous
nucleotides in the 5'-direction. In some embodiments, at least one
of the one or more groups is attached to one of the five 3'-most
nucleotides. In some embodiments, the group may be a label,
preferably a label which undergoes a detectable change in an
observable property upon becoming part of a double stranded
molecule, (e.g. by hybridizing to another nucleic acid molecule or
by nucleic acid synthesis or amplification). In some embodiments,
at least a portion of said oligonucleotide is hybridized to at
least a portion of said nucleic acid molecule. In some embodiments,
the oligonucleotide is capable of forming a hairpin. In some
embodiments, the oligonucleotide is in the form of a hairpin.
[0167] The present invention provides a method of making a
composition, comprising the steps of providing one or more
oligonucleotides and contacting the one or more oligonucleotides
with at least one nucleic acid molecule, wherein at least a portion
of at least one of said oligonucleotides is capable of hybridizing
with at least a portion of said nucleic acid molecule. Preferably,
the oligonucleotide comprises one or more specificity enhancing
groups and/or at least one detectable label. In some embodiments,
the group is a fluorescent moiety. A specificity enhancing group
may be attached at any position of the oligonucleotide that results
in the oligonucleotide being substantially less extendable when the
3'-most nucleotide of the oligonucleotide is not complementary to
the corresponding nucleotide of a target/template nucleic acid. In
some embodiments, at least one of the one or more groups is
attached to a nucleotide at or near the 3'-nucleotide. In some
embodiments, at least one of the one or more groups is attached to
one of the ten 3'-most nucleotides. In other words, in embodiments
of this type, at least one of the one or more specificity enhancing
groups may be attached to the 3'most nucleotide or any of the next
nine contiguous nucleotides in the 5'direction. In some
embodiments, at least one of the one or more groups is attached to
one of the five 3'-most nucleotides. In some embodiments, the group
may be a label, preferably a label which undergoes a detectable
change in an observable property upon becoming part of a double
stranded molecule, (e.g. by hybridizing to another nucleic acid
molecule). In some embodiments, at least a portion of said
oligonucleotide is hybridized to at least a portion of said nucleic
acid molecule. In some embodiments, the oligonucleotide is capable
of forming a hairpin. In some embodiments, the oligonucleotide is
in the form of a hairpin.
[0168] The present invention provides a method of determining the
presence of a particular nucleotide or nucleotides at a specific
position or positions in a target or template nucleic acid
molecule, comprising the steps of (a) contacting at least one
target or template nucleic acid molecule having a nucleotide or
nucleotides at a specific position or positions with one or more
oligonucleotides of the invention, wherein at least a portion of
the oligonucleotide is capable of forming base pairs (e.g.,
hybridizing) with at least a portion of the target or template
nucleic acid molecule said oligonucleotide preferably comprises at
least one specificity enhancing group and/or label; and (b)
incubating the oligonucleotide and the nucleic acid molecule
mixture under conditions sufficient to cause extension of the
oligonucleotide when the 3'-most nucleotide or nucleotides of the
oligonucleotide base pair with the nucleotide or nucleotides at the
specific position or positions of the nucleic acid target molecule.
Under such conditions, the production of an extension product
indicates the presence of the particular nucleotide or nucleotides
at the specific position or positions. In another aspect, the
invention provides a method for determining the absence of at least
one particular nucleotide at a specific position or positions in a
target or template nucleic acid molecule, comprising (a) contacting
at least one target nucleic acid molecule having a nucleotide or
nucleotides at a specific position with an oligonucleotide of the
invention, wherein at least a portion of the oligonucleotide is
capable of forming base pairs (e.g., hybridizing) with at least a
portion of the target nucleic acid molecule (said oligonucleotide
preferably comprising at least one specificity enhancing group or
label); and (b) incubating the oligonucleotide and the nucleic acid
molecule mixture under conditions sufficient to prevent or inhibit
extension of the oligonucleotide when the 3'-most nucleotide or
nucleotides of the oligonucleotide does not base pair (e.g., does
not hybridize) with the nucleotide at the specific position or
positions of the target nucleic acid molecule. Under such
conditions, the lack of production or reduced production of an
extension product indicates the absence of the particular
nucleotide or nucleotides at the specific position. In a preferred
aspect, the results of the extension of the oligonucleotide in the
above first method is compared to the lack or reduced level of
extension of the oligonucleotide in the above second method. In a
preferred aspect, the conditions in the first method are conducted
such that all or a portion of the target nucleic acid molecule is
amplified, while the conditions in the second method are conducted
such that the target nucleic acid molecule is not amplified or
amplified at a reduced level or slower rate compared to the
amplified target nucleic acid molecule produced by the first
method. In some embodiments, the specificity enhancing group is a
fluorescent moiety. A specificity enhancing group may be attached
at any position of the oligonucleotide that results in the
oligonucleotide being substantially less extendable when the
3'-most nucleotide of the oligonucleotide is not complementary to
the corresponding nucleotide of a target/template nucleic acid. In
some embodiments, at least one of the one or more groups is
attached to a nucleotide at or near the 3'-nucleotide. In some
embodiments, at least one of the one or more groups is attached to
one of the ten 3'-most nucleotides. In other words, in embodiments
of this type, at least one of the one or more specificity enhancing
groups may be attached to the 3'-most nucleotide or any of the next
nine contiguous nucleotides in the 5'-direction. In some
embodiments, at least one of the one or more groups is attached to
one of the five 3'-most nucleotides. In some embodiments, the group
may be a label, preferably a label which undergoes a detectable
change in an observable property upon becoming part of a double
stranded molecule, (e.g. by hybridizing to another nucleic acid
molecule). In some embodiments, at least a portion of said
oligonucleotide is hybridized to at least a portion of said nucleic
acid molecule. In some embodiments, the oligonucleotide is capable
of forming a hairpin. In some embodiments, the oligonucleotide is
in the form of a hairpin. The conditions of incubation preferably
include one or more polymerase enzymes such as Tsp DNA polymerase
(available from Life Technologies, Inc. Rockville Md.).
[0169] The present invention provides a method of synthesizing one
or more nucleic acid molecules, comprising (a) contacting at least
one target or template nucleic acid molecule with at least one
oligonucleotide of the invention, wherein at least a portion of
said oligonucleotide is capable of hybridizing with at least a
portion of said target/template nucleic acid molecule (said
oligonucleotide preferably comprises at least one specificity
enhancing group and/or label); and (b) incubating the target
nucleic acid and oligonucleotide mixture under conditions
sufficient to cause the extension of the oligonucleotide when the
3'-most nucleotide or nucleotides of the oligonucleotide are base
paired (e.g. hybridized) to said target nucleic acid molecule. In
another aspect, the invention provides a method for reduced
synthesis of one or more nucleic acid molecules, comprising (a)
contacting at least one target or template nucleic acid molecule
with at least one oligonucleotide of the invention, wherein at
least a portion of said oligonucleotide is capable of hybridizing
with at least a portion of said target/template nucleic acid
molecule (said oligonucleotide preferably comprises at least one
specificity enhancing group and/or label), and (b) incubating the
target/template nucleic acid molecule and oligonucleotide mixture
under conditions sufficient to prevent or inhibit extension of the
oligonucleotide when the 3'-most nucleotide or nucleotides of the
oligonucleotide does not base pair (e.g., does not hybridize) with
the nucleotide at the specific position or positions of the
target/template nucleic acid molecule. In a preferred aspect, the
results of the synthesis of the above first method is compared to
the lack or reduced level of synthesis in the above second method.
In a preferred aspect, the conditions of the first method are
conducted such that all or a portion of the target nucleic acid
molecule is amplified, while the conditions in the second method
are conducted such that a target nucleic acid molecule is not
amplified or amplified at a reduced level and/or a slower rate
compared to the amplified target nucleic acid molecule produced by
the first method. In some embodiments, the specificity enhancing
group is a fluorescent moiety. In some embodiments, the group is
attached to a nucleotide at or near the 3'-nucleotide. In some
embodiments, the group is attached to one of the ten 3'-most
nucleotides. In other words, in embodiments of this type, the group
may be attached to the 3'-most nucleotide or any of the next nine
contiguous nucleotides in the 5'-direction. In some embodiments,
the group may be a label, preferably a label which undergoes a
detectable change in an observable property upon becoming part of a
double stranded molecule, (e.g. by hybridizing to another nucleic
acid molecule). In some embodiments, at least a portion of said
oligonucleotide is hybridized to at least a portion of said nucleic
acid molecule. In some embodiments, the oligonucleotide is capable
of forming a hairpin. In some embodiments, the oligonucleotide is
in the form of a hairpin. The incubation conditions preferably
include one or more polymerase enzymes such as Tsp DNA polymerase
available from Life Technologies, Rockville, Md.
[0170] The present invention provides a method of quenching
fluorescence from a fluorescent moiety, comprising the step of
attaching the fluorescent moiety to an oligonucleotide, wherein the
oligonucleotide is capable of assuming a conformation in which the
oligonucleotide quenches the fluorescence of the fluorescent
moiety. In some embodiments, the conformation is a hairpin.
[0171] The present invention also relates to kits for the detection
or measurement of nucleic acid molecules or for polymerase activity
in a sample. Such kits may also be designed to detect/quantitate
nucleic acid molecules of interest during or after nucleic acid
synthesis or amplification reactions. Such kits may be diagnostic
kits where the presence of the nucleic acid is correlated with the
presence or absence of a disease or disorder. The invention also
relates to kits for carrying out extension, synthesis and/or
amplification reactions of the invention and to kits for making the
compositions of the invention.
[0172] In specific embodiments, the kits comprise one or more
oligonucleotides of the invention (including primers and/or
probes). The kit can further comprise additional components for
carrying out the detection/quantification assays or other methods
of the invention. Such kits may comprise one or more additional
components selected from the group consisting of one or more
polymerases (e.g., DNA polymerases and reverse transcriptases), one
or more nucleotides, one or more buffering salts (including nucleic
acid synthesis or amplification buffers), one or more control
nucleic acid target molecules (to act as positive controls to test
assay or assist in quantification of the amount of nucleic acid
molecules in unknown samples), one or more quenchers (single
stranded binding proteins, blocking oligonucleotides etc.),
instructions for carry one out the methods of the invention and the
like. Control nucleic acid molecules are preferably provided in the
kits of the invention at known concentrations to establish control
samples of known amounts of target molecules to assist one in
establishing the amount of nucleic acid molecule of interest in an
unknown sample. Thus, the measurement of activity of the labeled
oligonucleotide for a known sample may be compared to such
measurement for an unknown sample to quantify the amount of the
target nucleic acid molecule in the unknown sample. The kits of the
invention preferably comprise a container (a box, a carton, or
other packaging) having in close confinement therein one and
preferably more containers (tubes, vials and the like) which
comprise various reagents for carrying out the methods of the
invention. The reagents may be in separate containers or may be
combined in different combinations in a single container. Such kits
of the invention may further comprise instructions or protocols for
carrying out the methods of the invention and optionally may
comprise an apparatus or other equipment for detecting the
detectable labels associated with the oligonucleotides of the
invention.
[0173] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are obvious and may
be made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
Example 1
Preparation of Oligonucleotides
[0174] Oligonucleotides may be prepared using any known
methodology. In some preferred embodiments, oligonucleotides may be
synthesized on solid supports using commercially available
technology. Oligodeoxynucleotides were synthesized using DNA
synthesizer-8700 (Milligen/Biosearch). Fluorescent moieties may be
incorporated into the oligonucleotides of the present invention
using any conventional technology. For example, fluorescent labels
may be incorporated into nucleoside phosphoramidites and directly
incorporated into the oligodeoxynucleotides during automated
chemical synthesis. In some preferred embodiments, the modified
nucleotide may be a fluorescein-dT phosphoramidite (Glen Research
Cat #10-1056) which may be inserted into designated position during
chemical synthesis of oligonucleotide. 5'-fluorescein
phosphoramidite (FAM) (Glen Research Cat #10-5901) and 3'-TAMRA-CPG
500 (Glen Research cat #20-5910) were used to add the indicated
labels to the 5' and 3'-end respectively of the
oligodeoxynucleotide during chemical synthesis. Alternatively, a
nucleotide containing a reactive functional moiety may be
incorporated into the oligonucleotide during synthesis. After the
completion of the synthesis and removal of the oligonucleotide from
the solid support, the reactive functional moiety may be used to
couple a fluorescent moiety containing molecule to the
oligonucleotide. In some preferred embodiments, the reactive
functional moiety may be an amino-modified C6-dT (Glen Research
Catalog #10-1039) which may be inserted into designated position
during chemical synthesis of oligonucleotide and used for further
modification. The further modification may include the
incorporation of a fluorescently labeled molecule. In some
preferred embodiments, the fluorescently labeled molecule may be a
6-carboxyfluorescein succinimidyl ester (6-FAM, SE, cat# C6164
Molecular Probes), Fluorescein-5-isothiocyanate (FITC) (Molecular
probe cat# F-1907), 5-(6-)-carboxytetramethylrhodamine (TAMRA)
succinimidyl ester (Molecular Probes), or BODIPY 530/550
succinimidyl ester (Molecular Probes).
[0175] All labeled oligonucleotides may be purified using
reverse-phase HPLC, for example, on a C-18 column using a gradient
of acetonitrile in 0.2 M triethyl ammonium acetate.
[0176] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
Nucl. Acids Res. 16:3209 (1988), methylphosphonate oligonucleotides
can be prepared by use of controlled pore glass polymer supports
(Sarin et al., Proc. Natl. Acad. Sci. USA 85:7448-7451 (1988)).
Oligonucleotides may also be prepared by standard phosphoramidite
chemistry; or by cleavage of a larger nucleic acid fragment using
non-specific nucleic acid cleaving chemicals or enzymes or
site-specific restriction endonucleases. Labeled oligonucleotides
of the invention may also be obtained commercially from Life
Technologies, Inc. or other oligonucleotide manufactures.
[0177] A preferable method for synthesizing oligonucleotides is by
using an automated DNA synthesizer using methods known in the art.
Once the desired oligonucleotide is synthesized, it is cleaved from
the solid support on which it was synthesized and treated, by
methods known in the art, to remove any protecting groups present.
The oligonucleotide may then be purified by any method known in the
art, including extraction and gel purification. The concentration
and purity of the oligonucleotide may be determined by examining
the oligonucleotide that has been separated on an acrylamide gel,
or by measuring the optical density at 260 nm in a
spectrophotometer.
[0178] Oligonucleotides of the invention may be labeled during
chemical synthesis or the label may be attached after synthesis by
methods known in the art. In a specific embodiment, the label
moiety is a fluorophore. Suitable moieties that can be selected as
fluorophores or quenchers are set forth in Table 1.
TABLE-US-00001 TABLE 1
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid acridine
and derivatives: acridine acridine isothiocyanate
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)
4-amino-N-3-vinylsulfonyl)phenylnaphthalimide-3,5 disulfonate
(Lucifer Yellow VS) N-(4-anilino-1-naphthyl)maleimide
anthranilamide Brilliant Yellow coumarin and derivatives:
7-amino-4-methylcoumarin (AMC, Coumarin 120)
7-amino-4-trifluoromethylcouluarin (Coumaran 151) cyanosine
4',6-diaminidino-2-phenylindole (DAPI)
5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin
diethylenetriamine pentaacetate
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid
5-dimethylaminonaphthalene-1-sulfonyl chloride (DNS, dansyl
chloride) 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL)
4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC) eosin and
derivatives: eosin eosin isothiocyanate erythrosin and derivatives:
erythrosin B erythrosin isothiocyanate ethidium fluorescein and
derivatives: 5-carboxyfluorescein (FAM)
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE) fluorescein
fluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446
Malachite Green isothiocyanate 4-methylumbelliferone ortho
cresolphthalein nitrotyrosine pararosaniline Phenol Red
B-phycoerythrin o-phthaldialdehyde pyrene and derivatives: pyrene
pyrene butyrate succinimidyl 1-pyrene butyrate Reactive Red 4
(Cibacron .TM. Brilliant Red 3B-A) rhodamine and derivatives:
6-carboxy-X-rhodamine (ROX) 6-carboxyrhodamine (R6G) lissamine
rhodamine B sulfonyl chloride rhodamine (Rhod) rhodamine B
rhodamine 123 rhodamine X isothiocyanate sulforhodamine B
sulforhodamine 101 sulfonyl chloride derivative of sulforhodamine
101 (Texas Red) N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA)
tetramethyl rhodamine tetramethyl rhodamine isothiocyanate (TRITC)
riboflavin rosolic acid terbium chelate derivative
[0179] One of ordinary skill in the art can easily determine, using
art-known techniques of spectrophotometry, which of the above
identified fluorophores or combinations thereof can be used in
accordance with the invention. Oligonucleotides are preferably
modified during synthesis, such that a modified T-base is
introduced into a designated position by the use of Amino-Modifier
C6 dT (Glen Research), and a primary amino group is incorporated on
the modified T-base, as described by Ju et al. (Proc. Natl. Acad.
Sci., USA 92:4347-4351 (1995)). These modifications may be used for
subsequent incorporation of fluorescent dyes into designated
positions of the labeled oligonucleotides.
[0180] In yet another embodiment, the labeled oligonucleotides may
be further labeled with any other art-known detectable marker,
including radioactive labels such as .sup.32P, .sup.35S, .sup.3H,
and the like, or with enzymatic markers that produce detectable
signals when a particular chemical reaction is conducted, such as
alkaline phosphatase or horseradish peroxidase. Such enzymatic
markers are preferably heat stable, so as to survive the denaturing
steps of the amplification or synthesis process.
[0181] Oligonucleotides may also be indirectly labeled by
incorporating a nucleotide linked covalently to a hapten or to a
molecule such as biotin, to which a labeled avidin molecule may be
bound, or digoxygenin, to which a labeled anti-digoxygenin antibody
may be bound. Oligonucleotides may be supplementally labeled during
chemical synthesis or the supplemental label may be attached after
synthesis by methods known in the art.
[0182] The sequences of the primers used in the following specific
examples are provided in Table 2.
TABLE-US-00002 TABLE 2 Oligo A internally labeled with fluorescein
5'-cct tct cat ggtggc tgT aga ac (SEQ ID NO: 1) Oligo B 5'-labeled
with fluorescein 5'-Cct tct cat ggt ggc tgt aga ac (SEQ ID NO: 2)
Oligo C complement to oligo A and B 5'-gtt cta cag cca cca tga gaa
gg (SEQ ID NO: 3) Oligo D. 3'-labeled with TAMRA 5'-ggg get gcg act
gtg ctc cgg cA (SEQ ID NO: 4) Oligo E. complement to oligo D 5'-tgc
cgg agc aca gtc gca gcc cc (SEQ ID NO: 5) Oligo F. 5'-labeled with
fluorescein 5'-Aat aat agg atg agg cag ga (SEQ ID NO: 6) Oligo G.
5'-labeled with BODIPY 530/550 5'-Aat aat agg atg agg cag ga (SEQ
ID NO: 7) Oligo H complement to Oligo F and Oligo G 5'-tcc tgc ctc
atc cta tta tt (SEQ ID NO: 8) Oligo I forward primer for IL4 5'-gag
ttg acc gta aca gac atc tt (SEQ ID NO: 9 Oligo J. forward primer
for b-actin 5'-ggc att gcc gac agg aTg tag aag internally Labeled
with fluorescein (SEQ ID NO: 10) Oligo K. reverse primer for
b-actin 5'-ggg ccg gac tcg tca tac (SEQ ID NO: 11) Oligo L. forward
primer for b-actin labeled 5'-ggt tgT aga gca ctc agc aca atg aag a
with Fluorescein through the tail- (SEQ ID NO: 12) Oligo 1 IL 4
forward primer 5'-gag ttg acc gta aca gac atc tt (SEQ ID NO: 13)
Oligo 2 IL 4 reverse primer, 297WT 5'-cct tct cat ggt ggc tgt aga
ac (SEQ ID NO: 14) Oligo 3 IL 4 reverse primer, 297MUT 5'-cct tct
cat ggt ggc tgt aga at (SEQ ID NO: 15) Oligo 4 IL 4 reverse primer,
300WT 5'-gtg tcc ttc tca tgg tgg ctg tag (SEQ ID NO: 16) Oligo 5 IL
4 reverse primer, 300MUT 5'-gtg tcc ttc tca tgg tgg ctg tat (SEQ ID
NO: 17) Oligo 6 IL 4 reverse printer, 297WT-Fluo 5'-cct tct cat ggt
ggc tgT aga ac (SEQ ID NO: 18) Oligo 7 IL 4 reverse primer,
297MUT-Fluo 5'-cct tct cat ggt ggc tgT aga at (SEQ ID NO: 19) Oligo
8 IL 4 reverse primer, 300WT-Fluo 5'-gtg tcc ttc tca tgg tgg ctg
Tag (SEQ ID NO: 20) Oligo 9 IL 4 reverse primer, 300MUT-Fluo 5'-gtg
tcc ttc tca tgg tgg ctg Tat (SEQ ID NO: 21) Oligo 10 RDS reverse
primer-Fluo 5'-cta ccg ggt gtc tgt gtc tcg gTa g (SEQ ID NO: 22)
Oligo 11 RDS forward primer, C-allele 5'-cgt acc tgg cta tct gtg tc
(SEQ ID NO: 23) Oligo 12 RDS forward primer, T-allele 5'-cgt acc
tgg cta tct gtg tt (SEQ ID NO: 24) Oligo 13 RDS forward primer,
C-allele/hairpin 5'-gac acc tgg cta tct gtg tc (SEQ ID NO: 25)
Oligo 14 RDS forward primer, T-allele/hairpin 5'-aac aca cct ggc
tat ctg tgt t (SEQ ID NO: 26) Oligo 15 IL 4 reverse primer/hairpin
5'-cta cag tcc ttc tca tgg tgg ctg tag (SEQ ID NO: 27) Oligo 16
b-globin forward primer/linear-A 5'-ctt cct gag agc cga act gta gtg
a (SEQ ID NO: 28) Oligo 17 b-globin reverse primer/linear-A 5'-aca
tgt att tgc atg gaa aac aac tc (SEQ ID NO: 29) Oligo 18 b-globin
forward primer/hairpin-A 5'-tca cta ctt cct gag agc cga act gta gtg
a (SEQ ID NO: 30) Oligo 19 b-globin reverse primer/hairpin-A 5'-gag
ttg tac atg tat ttg cat gga aaa caa ctc (SEQ ID NO: 31) Oligo 20
b-globin forward primer/linear-B 5'-gct cag aat gat gtt tcc acc ttc
(SEQ ID NO: 32) Oligo 21 b-globin reverse primer/linear-B 5'-aaa
tca tac tag ctc acc agc aat g (SEQ ID NO: 33) Oligo 22 b-globin
forward primer/hairpin-B 5'-gaa ggt get cag aat gat gtt tcc acc ttc
(SEQ ID NO: 34) Oligo 23 b-globin reverse primer/hairpin-B 5'-cat
tgc aaa tca tac tag ctc acc agc aat g (SEQ ID NO: 35) Oligo 24 NF
1355 forward primer/linear 5'-tgg cag ttg aat gcc aag taa t (SEQ ID
NO: 36) Oligo 25 NF 1355 reverse primer/linear 5'-aca gcc act gtg
ccc agg tc (SEQ ID NO: 37) Oligo 26 NF 1355 forward primer/hairpin
5'-att act tgg cag ttg aat gcc aag taa t (SEQ ID NO: 38) Oligo 27
NF 1355 reverse primer/hairpin 5'-gac ctg aca gcc act gtg ccc agg
tc (SEQ ID NO: 39) Oligo 28 NF 1616 forward primer/linear 5'-att
tca tgg ggg aaa caa aga tg (SEQ ID NO: 40) Oligo 29 NF 1616 reverse
primer/linear 5'-ata cct gcg ctc acc aca gg (SEQ ID NO: 41) Oligo
30 NF 1616 forward primer/hairpin 5'-cat ctt tat ttc atg ggg gaa
aca aag atg (SEQ ID NO: 42) Oligo 31 NF 1616 reverse primer/hairpin
5'-cct gtg ata cct gcg ctc acc aca gg (SEQ ID NO: 43)
[0183] The nucleotide to which the fluorescent moiety is attached
is indicated by a bold capital letter.
Example 2
PCR Targets and Conditions
[0184] Those skilled in the art will appreciate that any nucleic
acid that can be amplified by PCR may be used in the practice of
the present invention. Examples of suitable nucleic acids include,
but are not limited to, genomic DNAs, cDNAs and cloned PCR
products. The practice of the present invention is not limited to
use with DNA molecules. For example, mRNA molecules may be used as
templates for an amplification reaction by first conducting a first
strand synthesis reaction using techniques well known in the art.
The present invention has been exemplified using cDNAs for IL4 and
b-actin synthesized using total mRNA from the corresponding cells
and SuperScript.TM. System for the First Strand cDNA Synthesis
(Gibco BRL, cat #18089-011) according to the manufacturer's manual.
IL4 and b-actin cDNAs were amplified and cloned into pTEPA plasmid
according to Gibco BRL manual (cat #10156-016).
[0185] The selection of suitable PCR conditions is within the
purview of ordinary skill in the art. Those skilled in the art will
appreciate that it may be necessary to adjust the concentrations of
the nucleic acid target, primers and temperatures of the various
steps in order to optimize the PCR reaction for a given target and
primer. Such optimization does not entail undue experimentation. In
the specific examples provided herein, PCR was performed in 25
.mu.l of PLATINUM.RTM. Taq Reaction Buffer with 0.5 un of
PLATINUM.RTM. Taq, 0.2 mM dNTPs, 0.2 .mu.M forward and reverse
primers, and 1.75 mM MgCl.sub.2 using 10.sup.4-10.sup.6 copies of
target. PLATINUM.RTM. Tsp was used under the same conditions.
Thermal cycling was performed on 9600 or ABI PRIZM.TM. 7700
Sequence Detector (Perkin Elmer) with 4 min denaturation at
94.degree. C., followed by 35-40 cycles: 15 sec at 94.degree. C.,
30 sec at 55.degree. C. and 40 sec at 72.degree. C. In two-step PCR
cycling conditions were 15 sec at 94.degree. C. and 30 sec at
65.degree. C.
Example 3
Detection of Nucleic Acids
[0186] Nucleic acids may be detected by any conventional
technology. In some preferred embodiments, the nucleic acid to be
detected may be a PCR product and may be detected either by agarose
gel electrophoresis or by homogeneous fluorescence detection method
as described in U.S. provisional patent application Ser. No.
60/139,890, filed Jun. 22, 1999. In this method fluorescent signal
is generated upon the incorporation of the specifically labeled
primer into the PCR product. The method does not require the
presence of any specific quenching moiety or detection
oligonucleotide. In some preferred embodiments, the detection
oligonucleotides are capable of forming a hairpin structure and are
labeled with fluorescein attached close to the 3'-end.
[0187] The fluorescent measurements were performed in the PCR
reaction buffer using on ABI PRIZM.TM. 7700 Sequence Detector,
fluorescent plate reader (TECAN) or Kodak EDAS Digital Camera.
Excitation/emission wavelengths were 490 nm/520 nm for fluorescein
and 555 nm/580 nm for TAMRA.
Example 4
Fluorescence Signal of Oligonucleotide Internally Labeled with
Fluorescein Increases Upon its Hybridization to the Complementary
Oligonucleotide
[0188] Two oligonucleotides of the same sequence were labeled with
fluorescein either internally on T-base (oligo A (SEQ ID NO:1)), or
at the 5'-end (oligo B (SEQ ID NO:2)) as described above. 10 pmoles
of each oligonucleotide was hybridized to the complementary oligo C
(SEQ ID NO:3) (50 pmoles) in 0.05 ml of the PCR buffer, heated at
70.degree. C. for 2 min and cooled to 25.degree. C. Melting curves
between 25 and 95.degree. C. were determined on ABI PRIZM.TM. 7700
Sequence Detector.
[0189] As shown in FIGS. 2A-2B, in case of internally labeled Oligo
A (SEQ ID NO:1), a fluorescence signal increases as a result of
presence of the non-labeled complementary oligonucleotide. That
means the signal increase was caused by the formation of the
double-stranded structure. In contrast, when the fluorescein was
present on the 5'-end of the same sequence (Oligo B (SEQ ID NO:2)),
fluorescence signal decreased upon hybridization.
Example 5
Oligodeoxynucleotide Labeled with TAMRA on its 3'-End, Increases
the Fluorescence Signal Upon Hybridization
[0190] 20 pmoles of Oligo D (SEQ ID NO:4) 3'-labeled with TAMRA as
described above was annealed to 100 pmoles of complementary
non-labeled oligodeoxynucleotide (Oligo E (SEQ ID NO:5)) in 0.5 ml
of the PCR Buffer. Fluorescence emission spectrum was detected on
spectrofluorimeter with 555 nm excitation.
[0191] As shown in FIG. 3, a significant increase of the signal was
observed upon hybridization, indicating that the proposed method
can be applied to different fluorophores. The curve labeled buffer
shows the fluorescence as a function of wavelength of the buffering
solution. The curve labeled single-stranded shows the results
obtained with the single-stranded version of oligo D (SEQ ID NO:4)
alone. When a non-complementary oligonucleotide was added to oligo
D (SEQ ID NO:4) a slight decrease in signal was observed
(+non-complement). When complementary oligonucleotide oligo E (SEQ
ID NO:5) was added, a large increase in fluorescence was observed
(+complement).
Example 6
Oligodeoxynucleotide 5'-Labeled with BODIPY 530/550 Increases the
Fluorescence Signal Upon Hybridization
[0192] In examples 4 and 5 oligonucleotides internally labeled with
fluorescein and 3' labeled with TAMRA were shown to increase the
fluorescence intensity upon hybridization to the complementary
oligonucleotide. In contrast, oligonucleotides 5'-labeled with
fluorescein demonstrated fluorescence quenching upon hybridization
(see example 4 and [Cardullo et al, 1988, PNAS 85, 8790-8794; Wu et
al. 1998, U.S. Pat. No. 5,846,729]).
[0193] However, there are some dyes that can show an enhancement of
the fluorescence intensity upon hybridization even though they are
located at the 5' position of an oligonucleotide. For example, an
oligodeoxynucleotide labeled at the 5' end with BODIPY 530/550
shows an increase fluorescence intensity upon hybridization.
[0194] The same oligodeoxynucleotide sequence was 5'-labeled with
fluorescein (Oligo F (SEQ ID NO:6)) or BODIPY 530/550 (Oligo G (SEQ
ID NO:7)). 20 pmoles of each labeled oligonucleotide was annealed
to 100 pmoles of complementary non-labeled oligodeoxynucleotide
(Oligo H (SEQ ID NO:8)) in 0.5 ml of the PCR Buffer. Fluorescence
emission spectrum was detected on spectrofluorimeter with 490 nm
excitation in case of fluorescein and 538 nm excitation in case of
BODIPY.
[0195] As shown in FIG. 4, a significant increase of the signal
upon hybridization in case of BODIPY dye was observed, in contrast,
a decrease in the signal was observed upon hybridization of a
fluorescein containing oligonucleotide.
[0196] The results shown in Examples 4, 5 and 6 demonstrate that
the fluorescent properties of a given fluorophore, in particular
the fluorescent intensity, can be affected upon hybridization
without significant shift of the emission spectrum as a result of
the point of attachment of the fluorphore to a given
oligonucleotide, i.e., internal, 3' and 5'.
Example 7
Quantitative PCR of IL4 cDNA Using Primer Internally Labeled with
Fluorescein
[0197] Fluorescein-dT was directly incorporated into the sequence
of IL-4 primer during chemical synthesis using the methods
described above. The resulting oligonucleotide (Oligo A (SEQ ID
NO:1)) was used as a reverse primer for IL4 cDNA amplification.
Quantitative PCR using reverse primer (Oligo A (SEQ ID NO:1)) and
forward primer (Oligo I (SEQ ID NO:9)) was performed as described
above in the presence of varying amounts of the template DNA.
10.sup.7, 10.sup.6, 10.sup.5, 10.sup.4, 10.sup.3, 10.sup.2, 10 and
0 copies of the cloned IL4 a target were used per reaction along
with four samples of unknown concentration of the target. As shown
in FIG. 5A, all dilutions of the DNA target can be detected with
extremely high accuracy.
[0198] The results of this experiment demonstrate that although no
quencher is present in the structure of labeled oligonucleotide, it
can be successfully used in quantitative PCR.
Example 8
Real-Time PCR of IL4 cDNA Using Primer Post-Synthetically Labeled
with FITC
[0199] Reverse primer for IL4 (Oligo A (SEQ ID NO:1)) was
synthesized and labeled post-synthetically as described above.
Amplification was performed with 10.sup.6, 10.sup.4, 10.sup.2 and 0
copies of nucleic acid target as described in the previous example.
As shown in FIG. 6, all dilutions of the DNA target can be
detected.
[0200] The experimental results in preceding examples demonstrate
that different methods of the labeling of oligonucleotides can be
used for achieving the same result. Also, since two methods of
synthesis provide different structures of the linker arm between
oligonucleotide and fluorophore, different linker arms can be used
to attach fluorophore in the proposed method.
Example 9
Real-Time PCR of b-Actin cDNA with a Primer Internally Labeled with
Fluorescein
[0201] Fluorescein-dT was directly incorporated into the sequence
of the forward primer for human b-actin cDNA (Oligo J (SEQ ID
NO:10)) during chemical synthesis. This oligonucleotide and
unlabeled reverse primer (Oligo K (SEQ ID NO:11)) were used for the
amplification of b-actin cDNA. cDNA target was obtained by reverse
transcription of HeLa cell mRNA and also a cloned cDNA fragment
(10.sup.7, 10.sup.5 and 0 copies per reaction). Quantitative PCR
was performed as described above. As shown in FIG. 7, all dilutions
of the DNA target can be detected.
[0202] The results of this experiment demonstrate that different
targets can be detected using the proposed method.
Example 10
Real-Time PCR of b-Actin cDNA with a Primer Internally Labeled
Through a "Tag" Sequence Non-Complementary to the Target
[0203] All the above experiments showed that the label could be
incorporated into the sequence of oligonucleotide complementary to
the target nucleic acid. However, the same result can be obtained
if the label is present on a non-complementary tag sequence
attached to the 5'-end of a PCR primer. In this case a signal will
be generated after this tailed primer is copied and incorporated
into the double-stranded PCR product. This approach was
demonstrated in the b-actin PCR.
[0204] Oligodeoxynucleotide (Oligo L (SEQ ID NO:12)) was
synthesized with Fluorescein-dT directly incorporated into the
structure of 9-nucleotide tail, non-complementary to the target.
This tail was added to the 5'-end of the b-actin forward primer.
Oligo L (SEQ ID NO:12) and unlabeled reverse primer (Oligo K (SEQ
ID NO:11)) were used to amplify b-actin cDNA and 10.sup.6,
10.sup.4, and 0 copies of cloned target. As shown in FIG. 8, both
cloned target and cDNA in total cDNA population can be
detected.
Example 11
Allele Specific PCR with Modified Oligonucleotide Primers
[0205] The principle of allele specific PCR is presented in FIG. 9.
The method operates on the basis of the specific amplification of a
target allele by the PCR with primers designed such that their 3'
ends are placed at the mutation site (i. e., the 3'-most nucleotide
of the primer corresponds to the mutated nucleotide in the
target/template nucleic acid). When this base is complementary to
that of the corresponding nucleotide of the specific allele, the
target is amplified; when it is not complementary PCR will proceed
with a significant delay. The longer the delay, the more
efficiently the system can discriminate between alleles. In some
preferred embodiments, the present invention provides
oligonucleotides useful for allele specific PCR which
oligonucleotides comprise a specificity enhancing group that
improves discrimination between alleles.
[0206] Allele specific PCR was performed using regular PCR primers
and the primers labeled with fluorescein at a base close to the
3'-end. Two positions of the IL4 cDNA were chosen for detection,
C297 and G300. For each position two PCRs were performed using the
same forward primer (Oligo 1 (SEQ ID NO:13)) and different reverse
primers: wild type (WT), complementary to the target, or mutant
(MUT) with a mismatch at the 3'-end. The sequences of the primers
used are provided in Table 2. Each of these allele specific primers
was synthesized with and without chemical modification on a T-base
close to the 3'-end. The primers used were 297 WT--primer
complementary to the C-allele at position 297 (Oligo 2 (SEQ ID
NO:14)), 297 MUT--same primer with C-T mutation at the 3'-end
(Oligo 3 (SEQ ID NO:15)), 300 WT--primer complementary to the
C-allele at position 300 (Oligo 4 (SEQ ID NO:16)) and 300 MUT--same
primer with G-T mutation at the 3'-end (Oligo 5 (SEQ ID NO:17)).
Oligonucleotides 6, 7, 8, 9 (SEQ ID NOs:18, 19, 20, 21,
respectively) correspond to oligonucleotides 2, 3, 4, 5 (SEQ ID
NOs:14, 15, 16, 17, respectively) with fluorescein attached to the
designated T-base.
[0207] Three step PCR was performed for 40 cycles with Platinum
Taq.TM. as described above and the results are shown in FIG. 10.
Reverse primers with their 3'-end at positions 297 or 300 were
either complementary to the target (WT) or had a 3' mutation (MUT).
Lanes 1 through 4 show the results obtained with primers modified
with fluorescein as a specificity enhancing group; lanes 5 through
8 show the results obtained with unmodified primers. Lanes 1 and 5
show the results using the primer 297 WT; lanes 2 and 6 show the
results using the primer 297 MUT; lanes 3 and 7 show the results
using primer 300 WT; lanes 4 and 8 show the results using primer
300 MUT. A comparison of lanes 2 and 6 and a comparison of lanes 4
and 8 show that the presence of a modification allows
discrimination that is almost complete after 40 cycles. The
practice of the present invention is not limited to the use of
fluorescein, similar results were obtained with TAMRA as a
specificity enhancing group (data not shown).
Example 12
Allele Specific PCR with Hairpin Oligonucleotide Primers
[0208] In some preferred embodiments, the primers of the present
invention may be modified such that they assume a hairpin
structure. This may be accomplished by adding one or more bases to
the 5'-terminal of the oligonucleotide which bases are selected to
be complementary to the bases at the 3'-terminal of the
oligonucleotide. In some preferred embodiments, at least one to
about 20 contiguous nucleotides are added to the 5' end of the
oligonucleotide that are complementary to the at least one to 20
contiguous nucleotides present in the 3'-end of the
oligonucleotide. In a preferred embodiment, from one to about 10
nucleotides are added to the 5'-end of the oligonucleotide, the
nucleotides selected such that they are complementary to the at
least one to about 10 contiguous nucleotides present in the 3'-end
of the oligonucleotide. In another preferred embodiment, from one
to about 5 nucleotides are added to the 5'-end of the
oligonucleotide, the nucleotides selected such that they are
complementary to the at least one to about 5 contiguous nucleotides
present in the 3'-end of the oligonucleotide.
[0209] The present invention is based upon the surprising result
that the mutation discrimination can be improved through the
secondary structure of the allele specific primers. This feature is
exemplified using primers specific for the RDS gene. Forward
primers for the RDS gene had their 3' ends located at position 558,
the site of a C/T polymorphism. The DNA target contained the
C-allele. The reverse primer was the same for both alleles and
contained the label that permitted homogeneous detection of
amplification in real time (Oligo 10 (SEQ ID NO:22)). Forward
allele specific primers were either of the conventional linear
structure (Oligo 11, 12 (SEQ ID NOs:23, 24, respectively)) or had
the hairpin structure (Oligo 13, 14 (SEQ ID NOs:25, 26,
respectively)). Hairpin primers consisted of the target-specific
sequence and a short tail complementary to the 3'-fragment of the
primer. Three step PCR was performed with Platinum Taq.TM. DNA
polymerase on PRIZM 7700 as described above. The results in FIG. 11
show that the blunt-end hairpin structure of the primer
significantly improves mutation discrimination. The primers of the
invention were used to discriminate between the C and the T allele
of human RDS gene by allele-specific PCR with Platinum Taq.TM. DNA
polymerase using the same fluorescent reverse primer (Oligo 10 (SEQ
ID NO:22)) and different allele specific forward primers. The
primers used were designated L-C for the linear primer specific for
C-allele (Oligo 11 (SEQ ID NO:23)), L-T for the linear primer
specific for T-allele (Oligo 12 (SEQ ID NO:24)), H-C for the
hairpin primer specific for C-allele (Oligo 13 (SEQ ID NO:25)) and
H-T for the hairpin primer specific for T-allele (Oligo 14 (SEQ ID
NO:26)). A comparison of the real time fluorescence of the
reactions is plotted as a function of the cycle number. The linear
T mismatched primer generated a signal that was detectable well
before the hairpin T mismatched primer signal. This demonstrates
that the discrimination between the alleles was improved by
incorporating the 3'-terminal of the primer into a hairpin
[0210] Another example of allele specific PCR using hairpin primers
is shown in FIG. 12. Here two genomic DNA samples were tested by
two step PCR. One of the samples was known to have a 558C-allele of
RDS gene, another the 558T allele. All forward primers were hairpin
primers and fluorescent reverse primer was used for the detection.
Curve 1 was obtained with the C-primer with C-target DNA; curve 2
was obtained using the C-primer with T-target DNA; curve 3 was
obtained using C-primer with no target DNA (negative control);
curve 4 was obtained using the T-primer with T-target DNA; curve 5
was obtained using T-primer with C-target DNA; curve 6 was obtained
using T-primer with no target (negative control).
[0211] The results demonstrate that only C-allele with C-specific
primers and T-allele with 1-specific primers gave a positive signal
when hairpin primers were used. No increase of fluorescence was
detected when the primer had a 3'-mismatch. No signal was generated
in the absence of target. As shown in FIGS. 13A-13B, the alleles
can be detected with the same high level of specificity not only in
real time but also at the end point. Fluorescent reverse primer was
used for the detection. 1, 3, 5 C-specific primers, 2, 4, 6
T-specific primers, 1 and 2 C allele target DNA, 3 and 4 T allele
target DNA, 5 and 6 no DNA (negative controls). Panel A shows a bar
graph of the fluorescence obtained while Panel B shows a photograph
of the reaction mixture after the amplification reactions. End
point detection is permitted by high signal/noise ratio of the
detection system and can be performed using fluorescent plate
reader or UV transilluminator and digital camera.
[0212] Another surprising result of the use of the primers of the
present invention is the elimination of primer dimers from the PCR
reaction. As shown in FIG. 14, the use of a hairpin oligonucleotide
in the PCR reaction eliminates the formation of primer dimers. IL4
cDNA was used as a PCR target. Oligo 1 (SEQ ID NO:13) was used as a
forward primer, oligo 2 (SEQ ID NO:14) as a linear reverse primer
and Oligo 15 (SEQ ID NO:27) as a hairpin reverse primer. PCR was
performed with platinum Taq.TM. for 50 cycles. Lanes 1, 5 contained
10.sup.6 copies of target; lanes 2, 6 contained 10.sup.4 copies of
target; lanes 3, 7 contained 10.sup.2 copies of target; and lanes
4, 8 contained no target. Comparison of the lanes 4 and 8 shows
that primer-dimer was formed with linear reverse primer but not
with the hairpin.
Example 13
Use of Mismatch Discriminating Polymerases in Allele Specific
PCR
[0213] The ability to discriminate between alleles by allele
specific PCR may be improved by using DNA polymerases modified to
be substantially unable to extend an oligonucleotide when the
3'-most nucleotide of the oligonucleotide is not base paired with
the target nucleic acid sequence. The preparation of such modified
DNA polymerases is disclosed in WO 99/10366 and WO 98/35060. These
publications disclose the cloning and mutagenesis of thermostable
polymerases, in particular, the thermostable DNA polymerase
isolated from Thermatoga spp. In some preferred embodiments of the
present invention, allele specific PCR is performed using a mutant
DNA polymerase derived from the DNA polymerase of Thermotoga
neopolitana (Tne). Suitable mutations include deletion of one or
more amino acids, frame shift mutations, point mutations that
result in one or more amino acid substitutions at one or more sites
in the enzyme, insertion mutations and combinations thereof. In a
preferred embodiment, the mutations may include a deletion of the
first 283 amino acids of the wild type enzyme leaving a fragment
that begins with methionine 284 (.DELTA.283), a point mutation
changing amino acid 323 from aspartic acid to alanine (D323A) and a
point mutation changing amino acid 722 from arginine to lysine
(R722K). In some preferred embodiments, the mutant Tne DNA
polymerase will have at least all three mutations, i.e. will be
.DELTA.283, D323A and R722K.
[0214] Platinum Tsp.TM. DNA polymerase is a proprietary enzyme of
LifeTechnologies that can be activated by temperature thus
providing a hot start for PCR (U.S. Pat. Nos. 5,338,671 and
5,587,287). Here we describe a new property of this enzyme,
increased specificity towards the base-paired 3'-end of the primer.
PCR was performed for 45 cycles with Platinum Tsp.TM. or Platinum
Taq.TM. DNA polymerase using IL4 cDNA as a target. Two positions of
the IL4 cDNA were chosen for detection, C297 and G300. For each
position two PCR reactions were performed using the same forward
primer (Oligo 1 (SEQ ID NO:13)) and different reverse primers.
Primer sequences are described in Table 1 (Oligos 1-5 (SEQ ID
NOs:13-17)). The oligonucleotides are designated wild type (WT),
when the 3'-nucleotide is complementary to the target, or mutant
(MUT) with a mismatch at the 3'-end. The oligonucleotides used were
the 297 WT primer which is complementary to the C-allele at
position 297 (Oligo 2 (SEQ ID NO:14), lane 1), the 297 MUT primer
which has the same sequence as the 297 WT primer except for a C-T
mutation at the 3'-end (Oligo 3 (SEQ ID NO:15), lane 3), the 300 WT
primer which is complementary to the C-allele at position 300
(Oligo 4 (SEQ ID NO:16), lane 2) and the 300 MUT primer which has
the same sequence as the 300 WT primer except for a G-T mutation at
the 3'-end (Oligo 5 (SEQ ID NO:17), lane 4). As seen in FIG. 15, a
comparison of the results obtained with Tsp.TM. DNA polymerase to
those obtained with Taq.TM. DNA polymerase show that Platinum
Tsp.TM. has better discriminatory properties than platinum
Taq.TM..
Example 14
Use of Hairpin Primers to Enhance Specificity of PCR
[0215] It has been unexpectedly found that the hairpin primers of
the present invention may be used to enhance the specificity of PCR
reactions. Without wishing to be bound by theory, it is believed
that the ability of the primers to form hairpin structures at
temperatures around the annealing temperature of the PCR reaction
makes the primers less capable of mis-priming to the target nucleic
acid molecule. This increase in specificity is not dependent upon
the particular target nucleic acid template and has been observed
with a variety of templates. The increase in specificity will be
particularly important for the amplification of templates that are
difficult to amplify and that produce low amounts or none of the
desired amplification product in PCR reactions.
[0216] In addition to hairpin structures, any structure that
sequesters the 3'-end of the oligonucleotide primer may be used to
practice the present invention. For example, the 5'-portion of the
primers of the present invention may be provided with sequence that
is capable of forming a duplex such that the 3'-end interacts with
the duplex to form a triplex. In general, any primer sequence that
reversibly involves the 3'-portion of the primer in a stable
structure that is not capable annealing to the template DNA while
in that structure may be used to practice the present invention. In
some embodiments, an oligonucleotide complementary to the primer
may be provided so as to sequester the 3'-end of the primer.
Complementary oligonucleotides may be provided with a
5'-overhanging region which may be designed to include self
complementary regions capable of forming hairpins. It is not
necessary that the entire 3'-portion of the primer be sequestered,
so long as the portion not sequestered is not capable of
mis-priming the nucleic acid template, it is sufficient to practice
the present invention.
[0217] In the first experiment, a 3.6 kb fragment of the human
beta-globin was amplified from human genomic DNA using Platinum Pfx
thermostable polymerase in Pfx buffer (LifeTechnologies). Two
different sets of primers were used. Each set of primers consisted
of two primer pairs, one pair of linear primers and another pair of
primers having a hairpin version of the same gene specific primer
sequence. The hairpin version of each pair of oligonucleotides was
constructed by adding bases to the 5'-end of the primer sequence
that are complementary to the 3'-end of the oligonucleotide.
Typically, the number of bases added to the 5'-end is selected such
that the oligonucleotide forms a hairpin at temperatures below the
annealing temperature and assumes a linear form at or near the
annealing temperature. Those skilled in the art can readily
determine the number of nucleotides to be added to the 5'-end of
the primer so as to control the temperature at which the primer
assumes a linear form. It is not necessary that the
oligonucleotides of the invention be entirely converted to linear
form at the annealing temperature; those skilled in the art will
appreciate that the oligonucleotides of the present invention may
be capable of reversibly melting and self reannealing (i.e.,
breathing) So long as the sequences of the oligonucleotides of the
invention are selected such that a sufficient number of
oligonucleotides are available to prime the extension/amplification
at the annealing temperature, the sequence is suitable for use in
the present invention whether or not some of the oligonucleotides
remain in a hairpin form at the annealing temperature. The number
of nucleotides that may be added may be from about 3 nucleotides to
about 25 nucleotides, or from about 3 nucleotides to about 20
nucleotides, or from about 3 nucleotides to about 15 nucleotides,
or from about 3 nucleotides to about 10 nucleotides, or from about
3 nucleotides to about 7 nucleotides. In some preferred
embodiments, from about 5 to about 8 nucleotides may be added to
the 5'-end of the primer oligonucleotide in order to form the
hairpin oligonucleotides of the present invention. For the
amplification of the beta globin gene, two sets of primers were
used. Set A oligos 16 (SEQ ID NO:28) and 17 (SEQ ID NO:29) (linear)
or 18 (SEQ ID NO:30) and 19 (SEQ ID NO:31) (hairpin) and Set
B--oligos 20 (SEQ ID NO:32) and 21 (SEQ ID NO:33) (linear) or 22
(SEQ ID NO:34) and 23 (SEQ ID NO:35) (hairpin). PCR was performed
as follows: 2 minutes at 94.degree. C. followed by 35 cycles of: 15
seconds at 94.degree. C. then 30 seconds at 60.degree. C. followed
by 4 minutes at 68.degree. C. using varying amounts of template
DNA. The results are shown in FIGS. 16A-16B. The lanes labeled M
contain molecular weight markers. Lanes 1 and 2 show the results
obtained using 50 ng of template DNA, lanes 3 and 4 show the
results obtained using 20 ng of template and lanes 5 and 6 show the
no DNA controls. It is clear that both linear sets of primers
generated various mis-priming products and primer-dimers, while
amplification with the corresponding hairpin primers produced the
expected size amplification product with very little incorrect
product.
[0218] Similar results were obtained during the amplification of
another human gene Necrosis Factor 2(NF2). 1.3 and 1.6 kb fragments
were amplified using Platinum Taq DNA polymerase in PCR SuperMix
(LifeTechnologies). For the amplification of the 1.3 kb fragment
oligos 24 (SEQ ID NO:36) and 25 (SEQ ID NO:37) (linear) or 26 (SEQ
ID NO:38) and 27 (SEQ ID NO:39) (hairpin) were used as primers. For
the amplification of the 1.6 kb fragment oligos 28 (SEQ ID NO:40)
and 29 (SEQ ID NO:41) (linear) or 30 (SEQ ID NO:42) and 31 (SEQ ID
NO:43) (hairpin) were used as primers. PCR was performed on 50 ng
of human genomic DNA as follows: 2 minutes at 94.degree. C.
followed by 35 cycles of: 30 seconds at 94.degree. C., 30 seconds
at 62.degree. C. and 4 minutes at 68.degree. C. The results are
shown in FIG. 17. Lane M contains molecular weight markers. +
indicates the presence of template DNA and - indicates the no DNA
control. Lane 1 shows the results using linear primers for the 1.3
kb fragment in the presence of template DNA. Lane 2 shows the no
DNA control for lane 1. Lane 3 shows the results obtained using the
hairpin primer for the 1.3 kb fragment while lane 4 is the no DNA
control for lane 3. Lane 5 shows the results obtained using the
linear primers for the 1.6 kb fragment while lane 6 is the no DNA
control for lane 5. Lane 7 shows the results obtained using the
hairpin primers for the 1.6 kb fragment while lane 8 is the no DNA
control for lane 7. In both instances the hairpin primers gave more
and cleaner amplification products of the appropriate size than
linear primers of the same gene specific sequence.
[0219] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0220] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
43123DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 1ccttctcatg gtggctgtag aac 23223DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
2ccttctcatg gtggctgtag aac 23323DNAArtificial SequenceDescription
of Artificial Sequence Oligonucleotide 3gttctacagc caccatgaga agg
23423DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 4ggggctgcga ctgtgctccg gca 23523DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
5tgccggagca cagtcgcagc ccc 23620DNAArtificial SequenceDescription
of Artificial Sequence Oligonucleotide 6aataatagga tgaggcagga
20720DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 7aataatagga tgaggcagga 20820DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
8tcctgcctca tcctattatt 20923DNAArtificial SequenceDescription of
Artificial Sequence Oligonucleotide 9gagttgaccg taacagacat ctt
231024DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 10ggcattgccg acaggatgta gaag 241118DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
11gggccggact cgtcatac 181228DNAArtificial SequenceDescription of
Artificial Sequence Oligonucleotide 12ggttgtagag cactcagcac
aatgaaga 281323DNAArtificial SequenceDescription of Artificial
Sequence Oligonucleotide 13gagttgaccg taacagacat ctt
231423DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 14ccttctcatg gtggctgtag aac 231523DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
15ccttctcatg gtggctgtag aat 231624DNAArtificial SequenceDescription
of Artificial Sequence Oligonucleotide 16gtgtccttct catggtggct gtag
241724DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 17gtgtccttct catggtggct gtat 241823DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
18ccttctcatg gtggctgtag aac 231923DNAArtificial SequenceDescription
of Artificial Sequence Oligonucleotide 19ccttctcatg gtggctgtag aat
232024DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 20gtgtccttct catggtggct gtag 242124DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
21gtgtccttct catggtggct gtat 242225DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
22ctaccgggtg tctgtgtctc ggtag 252320DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
23cgtacctggc tatctgtgtc 202420DNAArtificial SequenceDescription of
Artificial Sequence Oligonucleotide 24cgtacctggc tatctgtgtt
202520DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 25gacacctggc tatctgtgtc 202622DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
26aacacacctg gctatctgtg tt 222727DNAArtificial SequenceDescription
of Artificial Sequence Oligonucleotide 27ctacagtcct tctcatggtg
gctgtag 272825DNAArtificial SequenceDescription of Artificial
Sequence Oligonucleotide 28cttcctgaga gccgaactgt agtga
252926DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 29acatgtattt gcatggaaaa caactc 263031DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
30tcactacttc ctgagagccg aactgtagtg a 313133DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
31gagttgtaca tgtatttgca tggaaaacaa ctc 333224DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
32gctcagaatg atgtttccac cttc 243325DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
33aaatcatact agctcaccag caatg 253430DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
34gaaggtgctc agaatgatgt ttccaccttc 303531DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
35cattgcaaat catactagct caccagcaat g 313622DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
36tggcagttga atgccaagta at 223720DNAArtificial SequenceDescription
of Artificial Sequence Oligonucleotide 37acagccactg tgcccaggtc
203828DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 38attacttggc agttgaatgc caagtaat
283926DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 39gacctgacag ccactgtgcc caggtc 264023DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
40atttcatggg ggaaacaaag atg 234120DNAArtificial SequenceDescription
of Artificial Sequence Oligonucleotide 41atacctgcgc tcaccacagg
204230DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 42catctttatt tcatggggga aacaaagatg
304326DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 43cctgtgatac ctgcgctcac cacagg 26
* * * * *