FIELD
[0003] The methods and compositions described herein relate generally to the
area of nucleic acid amplification. In particular, described herein are methods and
compositions for increasing amplification efficiency.
BACKGROUND
15 [0004] A wide variety of nucleic acid amplification methods are available, and
many have been employed in the implementation of sensitive diagnostic assays based on
nucleic acid detection. Polymerase chain reaction (PCR) remains the most widely used
DNA amplification and quantitation method. Nested PCR, a two-stage PCR, is used to
increase the specificity and sensitivity of the PCR (U.S. Patent No. 4,683,195). Nested
20 primers for use in the PCR amplification are oligonucleotides having sequence
complementary to a region on a target sequence between reverse and forward primer
targeting sites. However, PCR in general has several limitations. Standard PCR
amplification can only achieve less than two-fold increase of the amount of target
sequence at each cycle. It is still relatively slow. In addition, the sensitivity of this
25 method is typically limited, making it difficult to detect target that may be present at only
a few molecules in a single reaction.
[0005] "Catalytic hybridization amplification" (CHA), alternatively known as
"cycling probe technology," is described in PCT publication no. WO 89/09284, and U.S.
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Patent Nos. 5,011,769 and 4,876,187. Briefly, CHA is an improved hybridization assay
method whereby the target sequence to be detected is able to capture many molecules of
the probe in a repeating series of reactions (i.e., "cycling probe"). Essentially, enzymemediated
cleavage of the probe within the probe target duplex results in release of the
5 intact target sequence, which can repeatedly recycle through the reaction pathway. The
target sequence serves as a catalytic cofactor for the cleavage of a complementary,
labeled nucleic acid probe that is hybridized to the target. The detectable signal in this
reaction results from cleavage of the probe, e.g., after repeated CHA cycles, one
measures the labeled probe cleavage product. The CHA method is useful in detecting
10 specific DNA or RNA sequences.
SUMMARY
[0006] Various embodiments contemplated herein may include, but need not be
limited to, one or more of the following:
[0007] Embodiment 1: A nucleic acid primer set for amplifying a target nucleic
15 acid in a sample, wherein the target nucleic acid includes a first template strand and,
optionally, a second template strand, wherein the second template strand is
complementary to the first template strand, the primer set including a cycling probe and
oligonucleotides in the form of, or capable of forming, at least two first primers capable
of hybridizing to the first template strand, wherein the at least two first primers comprise
20 a first outer primer and a first inner primer,
[0008] the first outer primer including a primer sequence a that specifically
hybridizes to first template strand sequence a', primer sequence a including one or more
first modified base(s); and
[0009] the first inner primer including a single-stranded primer sequence b that
25 specifically hybridizes to first template strand sequence b', wherein b' is adjacent to, and
5' of, a', and wherein single-stranded primer sequence b is linked at its 5' end to a first
strand of a double-stranded primer sequence including:
[0010] a primer sequence a adjacent to, and 5' of, single-stranded primer
sequence b; and
30 [0011] a clamp sequence c adjacent to, and 5' of, primer sequence a,
wherein clamp sequence cis not complementary to a first strand template sequence d',
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which is adjacent to, and 3' of, first strand template sequence a'; wherein a second strand
of the double-stranded primer sequence includes primer sequence c' adjacent to, and 3'
of, primer sequence a', wherein combined sequence c' -a' is complementary to combined
sequence c-a, primer sequence a' including one or more second modified base(s); and
5 wherein the unmodified forms of the first and second modified bases are complementary,
and the first and second modified bases preferentially pair with the unmodified forms, as
compared to pairing between the first and second modified bases.
[0012] Embodiment 2: The primer set of embodiment 1, wherein the primer set
additionally includes at least one second primer capable of specifically hybridizing to the
10 second template strand.
15
[0013] Embodiment 3: A method for amplifying a target nucleic acid in a
sample, wherein the target nucleic acid includes a first template strand and, optionally, a
second template strand, wherein the second template strand is complementary to the first
template strand, the method including:
[0014]
[0015]
(a) contacting the sample with a cycling probe including a label and:
(i) at least two first primers capable of hybridizing to the first
template strand, wherein the at least two first primers comprise a first outer primer and a
first inner primer,
[0016] the first outer primer including a primer sequence a that
20 specifically hybridizes to first template strand sequence a', primer sequence a including
one or more first modified base(s); and
[0017] the first inner primer including a single-stranded primer
sequence b that specifically hybridizes to first template strand sequence b', wherein b' is
adjacent to, and 5' of, a', and wherein single-stranded primer sequence b is linked at its
25 5' end to a first strand of a double-stranded primer sequence including:
[0018] a primer sequence a adjacent to, and 5' of, singlestranded
primer sequence b; and
[0019] a clamp sequence c adjacent to, and 5' of, primer
sequence a, wherein clamp sequence c is not complementary to a first strand template
30 sequenced', which is adjacent to, and 3' of, first strand template sequence a'; wherein a
second strand of the double-stranded primer sequence includes primer sequence c'
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adjacent to, and 3' of, primer sequence a', wherein combined sequence c' -a' is
complementary to combined sequence c-a, primer sequence a' including one or more
second modified base(s); wherein the unmodified forms of the first and second modified
bases are complementary, and the first and second modified bases preferentially pair
5 with the unmodified forms, as compared to pairing between the first and second
modified bases; and
10
15
20
[0020] (ii) at least one second primer capable of specifically
hybridizing to the second template strand, wherein the contacting is carried out under
conditions wherein the primers anneal to their template strands, if present;
[0021] (b) amplifying the target nucleic acid, if present, using a DNA
polymerase lacking 5' -3' exonuclease activity, under conditions where strand
displacement occurs, to produce amplicons that comprise sequence extending from
template sequence a' to the binding site for the second primer; and
[0022]
[0023]
(c) detecting, and optionally quantifying, the target nucleic acid.
Embodiment 4: The method of embodiment 3, wherein the DNA
polymerase is stable above 85 degrees centigrade.
[0024] Embodiment 5: The primer set or method of any one of the preceding
embodiments, wherein the T m of combined sequence c-a, in double-stranded form, is
greater than that of combined sequence a-b, in double-stranded form.
[0025] Embodiment 6: The primer set or method of any one of the preceding
embodiments, wherein combined sequence c-a is more GC-rich than combined sequence
a-b, and/or contains more stabilizing bases.
[0026] Embodiment 7: The primer set or method of any one of the preceding
embodiments, wherein the primer set is capable of amplifying, or the method amplifies,
25 the target nucleic acid at the rate of at least 3number of cycles during an exponential phase of
amplification.
[0027] Embodiment 8: The primer set or method of any one of the preceding
embodiments, wherein the primer set or method permits detection of a single-copy
nucleic acid in a biological sample within about 12% - 42% fewer amplification cycles
30 than would be required for said detection using only a single forward and a single reverse
primer.
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[0028] Embodiment 9: The primer set or method of any one of embodiments 2-
8, wherein the second primer includes oligonucleotides in the form of, or capable of
forming, at least two second primers capable of hybridizing to the second template
strand, wherein the at least two second primers comprise a second outer primer and a
5 second inner primer,
[0029] the second outer primer including a primer sequence e that specifically
hybridizes to second template strand sequence e', primer sequence e including one or
more third modified base(s); and
[0030] the second inner primer including a single-stranded primer sequence f that
10 specifically hybridizes to second template strand sequence f', wherein f' is adjacent to,
and 5' of, e', and wherein single-stranded primer sequence f is linked at its 5' end to a
first strand of a double-stranded primer sequence including:
15
[0031]
sequence f; and
[0032]
a primer sequence e adjacent to, and 5' of, single-stranded primer
a clamp sequence g adjacent to, and 5' of, primer sequence e,
wherein clamp sequence g is not complementary to second strand template sequence h',
which is adjacent to, and 3', of second template strand sequence e'; wherein a second
strand of the double-stranded primer sequence includes primer sequence g' adjacent to,
and 3' of, primer sequence e', wherein combined sequence g' -e' is complementary to
20 combined sequence g-e, primer sequence e' including one or more fourth modified
base(s); and wherein the unmodified forms of the third and fourth modified bases are
complementary, and the third and fourth modified bases preferentially pair with the
unmodified forms, as compared to pairing between the third and fourth modified bases.
[0033] Embodiment 10: The primer set or method of embodiment 9, wherein the
25 T m of combined sequence g-e, in double-stranded form is greater than that of combined
sequence e-f, in double-stranded form.
30
[0034] Embodiment 11: The primer set or method of any one of embodiments 9-
10, wherein combined sequence g-e is more GC-rich than combined sequence e-f, and/or
contains more stabilizing bases.
[0035] Embodiment 12: The primer set or method of any one of embodiments 9-
11, wherein the primer set is capable of amplifying, or the method amplifies, the target
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nucleic acid at the rate of at least 6number of cycles during an exponential phase of
amplification.
[0036] Embodiment 13: The primer set or method of any one of embodiments 9-
12, wherein the primer set or method permits detection of a single-copy nucleic acid in a
5 biological sample within about 36% - 66% fewer amplification cycles than would be
required for said detection using only a single forward and a single reverse primer.
[0037] Embodiment 14: The primer set or method of any one of the preceding
embodiments, wherein clamp sequence(s) c and g, if present, is/are not capable of being
copied during amplification.
10 [0038] Embodiment 15: The primer set or method of embodiment 14, wherein
clamp sequence(s) c and/or g, if present, comprise(s) 2' -0-methyl RNA.
15
[0039] Embodiment 16: The primer set or method of any one of the preceding
embodiments, wherein the double-stranded primer sequence of the first inner primer
and/or the second inner primer, if present, does not comprise a hairpin sequence.
[0040] Embodiment 17: The primer set or method of any one of embodiments 1-
15, wherein the double-stranded primer sequence of the first inner primer includes a
hairpin sequence in which clamp sequence c is linked to complementary sequence c'
and/or the double-stranded primer sequence of the second inner primer, if present,
includes a hairpin sequence in which clamp sequence g is linked to complementary
20 sequence g'.
[0041] Embodiment 18: A nucleic acid primer set for amplifying a target nucleic
acid in a sample, wherein the target nucleic acid includes a first template strand and,
optionally, a second template strand, wherein the second template strand is
complementary to the first template strand, the primer set including a cycling probe
25 including a label and oligonucleotides in the form of, or capable of forming, at least three
first primers capable of hybridizing to the first template strand, wherein the at least three
first primers comprise a first outer primer, a first intermediate primer, and a first inner
primer,
[0042] the first outer primer including a primer sequence d that specifically
30 hybridizes to first template strand sequence d', primer sequence d including one or more
first modified base(s);
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[0043] the first intermediate primer including a single-stranded primer sequence
a that specifically hybridizes to first template strand sequence a', wherein a' is adjacent
to, and 5' of, d', primer sequence a including one or more second modified base(s),
wherein single-stranded primer sequence a is linked at its 5' end to a first strand of a
5 double-stranded primer sequence including:
[0044]
sequence a; and
[0045]
a primer sequenced adjacent to, and 5' of, single-stranded primer
a clamp sequence c1 adjacent to, and 5' of, primer sequenced,
wherein clamp sequence c1 is not complementary to a first template strand sequence i',
10 which is adjacent to, and 3' of, first template strand sequenced'; wherein a second strand
of the double-stranded primer sequence includes primer sequence c1' adjacent to, and 3'
of, primer sequenced', wherein combined sequence cl'-d' is complementary to
combined sequence cl-d, primer sequenced' including one or more third modified
15
base( s); and
[0046] the first inner primer including a single-stranded primer sequence b that
specifically hybridizes to first template strand sequence b', wherein b' is adjacent to, and
5' of, a', and wherein single-stranded primer sequence b is linked at its 5' end to a first
strand of a double-stranded primer sequence including:
[0047] a primer sequence a adjacent to, and 5' of, single-stranded primer
20 sequence b;
[0048]
[0049]
a primer sequenced adjacent to, and 5' of, primer sequence a; and
a clamp sequence c2 adjacent to, and 5' of, primer sequenced,
wherein clamp sequence c2 is not complementary to first strand template sequence i';
wherein a second strand of the double-stranded primer sequence of the inner primer
25 includes primer sequence c2' adjacent to, and 3' of, primer sequenced', which is
adjacent to, and 3' of, primer sequence a', primer sequence a' including one or more
fourth modified base(s), wherein combined sequence c2'-d'-a' is complementary to
combined sequence c2-d-a; wherein the unmodified forms of the first and third modified
bases are complementary, and the first and third modified bases preferentially pair with
30 the unmodified forms, as compared to pairing between the first and third modified bases;
and wherein the unmodified forms of the second and fourth modified bases are
complementary, and the second and fourth modified bases preferentially pair with the
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unmodified forms, as compared to pairing between the second and fourth modified
bases.
[0050] Embodiment 19: The primer set of embodiment 18, wherein the primer
set additionally includes at least one second primer capable of specifically hybridizing to
5 the second template strand.
10
[0051] Embodiment 20: A method for amplifying a target nucleic acid in a
sample, wherein the target nucleic acid includes a first template strand and, optionally, a
second template strand, wherein the second template strand, if present is complementary
to the first template strand, the method including:
[0052]
[0053]
(a) contacting the sample with a cycling probe including a label and:
(i) at least three first primers capable of hybridizing to the
first template strand, wherein the at least three first primers comprise a first outer primer,
a first intermediate primer, and a first inner primer,
[0054] the first outer primer including a primer sequence d that
15 specifically hybridizes to first template strand sequenced', primer sequenced including
one or more first modified base(s);
[0055] the first intermediate primer including a single-stranded
primer sequence a that specifically hybridizes to first template strand sequence a',
wherein a' is adjacent to, and 5' of, d', primer sequence a including one or more second
20 modified base(s), wherein single-stranded primer sequence a is linked at its 5' end to a
first strand of a double-stranded primer sequence including:

CLAIMS
What is claimed is:
1. A nucleic acid primer set for amplifying a target nucleic acid in a sample,
wherein the target nucleic acid comprises a first template strand and, optionally, a second
5 template strand, wherein the second template strand is complementary to the first
template strand, the primer set comprising a cycling probe and oligonucleotides in the
form of, or capable of forming, at least two first primers capable of hybridizing to the
first template strand, wherein the at least two first primers comprise a first outer primer
and a first inner primer,
10 the first outer primer comprising a primer sequence a that specifically hybridizes
to first template strand sequence a', primer sequence a comprising one or more first
modified base(s); and
the first inner primer comprising a single-stranded primer sequence b that
specifically hybridizes to first template strand sequence b', wherein b' is adjacent to, and
15 5' of, a', and wherein single-stranded primer sequence b is linked at its 5' end to a first
strand of a double-stranded primer sequence comprising:
a primer sequence a adjacent to, and 5' of, single-stranded primer
sequence b; and
a clamp sequence c adjacent to, and 5' of, primer sequence a, wherein
20 clamp sequence cis not complementary to a first strand template sequenced', which is
adjacent to, and 3' of, first strand template sequence a';
wherein a second strand of the double-stranded primer sequence comprises
primer sequence c' adjacent to, and 3' of, primer sequence a', wherein combined
sequence c'-a' is complementary to combined sequence c-a, primer sequence a'
25 comprising one or more second modified base(s); and
wherein the unmodified forms of the first and second modified bases are
complementary, and the first and second modified bases preferentially pair with the
unmodified forms, as compared to pairing between the first and second modified bases.
2. The primer set of claim 1, wherein the primer set additionally comprises at least
30 one second primer capable of specifically hybridizing to the second template strand.
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3. A method for amplifying a target nucleic acid in a sample, wherein the target
nucleic acid comprises a first template strand and, optionally, a second template strand,
wherein the second template strand is complementary to the first template strand, the
method comprising:
5 (a) contacting the sample with a cycling probe comprising a label and:
(i) at least two first primers capable of hybridizing to the first
template strand, wherein the at least two first primers comprise a first outer primer and a
first inner primer,
the first outer primer comprising a primer sequence a that
10 specifically hybridizes to first template strand sequence a', primer sequence a
comprising one or more first modified base(s); and
the first inner primer comprising a single-stranded primer
sequence b that specifically hybridizes to first template strand sequence b', wherein b' is
adjacent to, and 5' of, a', and wherein single-stranded primer sequence b is linked at its
15 5' end to a first strand of a double-stranded primer sequence comprising:
a primer sequence a adjacent to, and 5' of, single-stranded
primer sequence b; and
a clamp sequence c adjacent to, and 5' of, primer sequence
a, wherein clamp sequence c is not complementary to a first strand template sequence d',
20 which is adjacent to, and 3' of, first strand template sequence a';
25
wherein a second strand of the double-stranded primer sequence
comprises primer sequence c' adjacent to, and 3' of, primer sequence a', wherein
combined sequence c'-a' is complementary to combined sequence c-a, primer sequence
a' comprising one or more second modified base(s);
wherein the unmodified forms of the first and second modified
bases are complementary, and the first and second modified bases preferentially pair
with the unmodified forms, as compared to pairing between the first and second
modified bases; and
(ii) at least one second primer capable of specifically hybridizing to
30 the second template strand,
wherein the contacting is carried out under conditions wherein the primers
anneal to their template strands, if present;
(b) amplifying the target nucleic acid, if present, using a DNA polymerase
lacking 5' -3' exonuclease activity, under conditions where strand displacement occurs,
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to produce amplicons that comprise sequence extending from template sequence a' to the
binding site for the second primer; and
(c) detecting, and optionally quantifying, the target nucleic acid.
4. The primer set or method of any one of the preceding claims, wherein the T m of
5 combined sequence c-a, in double-stranded form, is greater than that of combined
sequence a-b, in double-stranded form.
5. The primer set or method of any one of claims 2-4, wherein the second primer
comprises oligonucleotides in the form of, or capable of forming, at least two second
primers capable of hybridizing to the second template strand, wherein the at least two
10 second primers comprise a second outer primer and a second inner primer,
the second outer primer comprising a primer sequence e that specifically
hybridizes to second template strand sequence e', primer sequence e comprising one or
more third modified base(s); and
the second inner primer comprising a single-stranded primer sequence f that
15 specifically hybridizes to second template strand sequence f', wherein f' is adjacent to,
and 5' of, e', and wherein single-stranded primer sequence f is linked at its 5' end to a
first strand of a double-stranded primer sequence comprising:
a primer sequence e adjacent to, and 5' of, single-stranded primer
sequence f; and
20 a clamp sequence g adjacent to, and 5' of, primer sequence e, wherein
clamp sequence g is not complementary to second strand template sequence h', which is
adjacent to, and 3', of second template strand sequence e';
wherein a second strand of the double-stranded primer sequence comprises
primer sequence g' adjacent to, and 3' of, primer sequence e', wherein combined
25 sequence g' -e' is complementary to combined sequence g-e, primer sequence e'
comprising one or more fourth modified base(s); and
30
wherein the unmodified forms of the third and fourth modified bases are
complementary, and the third and fourth modified bases preferentially pair with the
unmodified forms, as compared to pairing between the third and fourth modified bases.
6. The primer set or method of claim 5, wherein the Tm of combined sequence g-e,
in double-stranded form is greater than that of combined sequence e-f, in doublestranded
form.
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5
7. The primer set or method of any one of the preceding claims, wherein clamp
sequence(s) c and g, if present, is/are not capable of being copied during amplification.
8. The primer set or method of claim 7, wherein clamp sequence(s) c and/or g, if
present, comprise(s) 2' -0-methyl RNA.
9. A nucleic acid primer set for amplifying a target nucleic acid in a sample,
wherein the target nucleic acid comprises a first template strand and, optionally, a second
template strand, wherein the second template strand is complementary to the first
template strand, the primer set comprising a cycling probe comprising a label and
oligonucleotides in the form of, or capable of forming, at least three first primers capable
10 of hybridizing to the first template strand, wherein the at least three first primers
comprise a first outer primer, a first intermediate primer, and a first inner primer,
15
the first outer primer comprising a primer sequence d that specifically hybridizes
to first template strand sequenced', primer sequenced comprising one or more first
modified base(s);
the first intermediate primer comprising a single-stranded primer sequence a that
specifically hybridizes to first template strand sequence a', wherein a' is adjacent to, and
5' of, d', primer sequence a comprising one or more second modified base(s), wherein
single-stranded primer sequence a is linked at its 5' end to a first strand of a doublestranded
primer sequence comprising:
20 a primer sequenced adjacent to, and 5' of, single-stranded primer
25
sequence a; and
a clamp sequence c1 adjacent to, and 5' of, primer sequenced, wherein
clamp sequence c1 is not complementary to a first template strand sequence i', which is
adjacent to, and 3' of, first template strand sequenced';
wherein a second strand of the double-stranded primer sequence
comprises primer sequence cl' adjacent to, and 3' of, primer sequenced', wherein
combined sequence cl'-d' is complementary to combined sequence cl-d, primer
sequenced' comprising one or more third modified base(s); and
the first inner primer comprising a single-stranded primer sequence b that
30 specifically hybridizes to first template strand sequence b', wherein b' is adjacent to, and
5' of, a', and wherein single-stranded primer sequence b is linked at its 5' end to a first
strand of a double-stranded primer sequence comprising:
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a primer sequence a adjacent to, and 5' of, single-stranded primer
sequence b;
a primer sequenced adjacent to, and 5' of, primer sequence a; and
a clamp sequence c2 adjacent to, and 5' of, primer sequenced, wherein
5 clamp sequence c2 is not complementary to first strand template sequence i';
wherein a second strand of the double-stranded primer sequence of the
inner primer comprises primer sequence c2' adjacent to, and 3' of, primer sequenced',
which is adjacent to, and 3' of, primer sequence a', primer sequence a' comprising one
or more fourth modified base(s), wherein combined sequence c2'-d'-a' is
10 complementary to combined sequence c2-d-a;
wherein the unmodified forms of the first and third modified bases are
complementary, and the first and third modified bases preferentially pair with the
unmodified forms, as compared to pairing between the first and third modified bases;
and
15 wherein the unmodified forms of the second and fourth modified bases are
complementary, and the second and fourth modified bases preferentially pair with the
unmodified forms, as compared to pairing between the second and fourth modified
bases.
10. The primer set of claim 9, wherein the primer set additionally comprises at least
20 one second primer capable of specifically hybridizing to the second template strand.
11. A method for amplifying a target nucleic acid in a sample, wherein the target
nucleic acid comprises a first template strand and, optionally, a second template strand,
wherein the second template strand, if present is complementary to the first template
strand, the method comprising:
25 (a) contacting the sample with a cycling probe comprising a label and:
(i) at least three first primers capable of hybridizing to the first
template strand, wherein the at least three first primers comprise a first outer primer, a
first intermediate primer, and a first inner primer,
the first outer primer comprising a primer sequence d that
30 specifically hybridizes to first template strand sequence d', primer sequenced
comprising one or more first modified base(s);
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the first intermediate primer comprising a single-stranded primer
sequence a that specifically hybridizes to first template strand sequence a', wherein a' is
adjacent to, and 5' of, d', primer sequence a comprising one or more second modified
base(s), wherein single-stranded primer sequence a is linked at its 5' end to a first strand
5 of a double-stranded primer sequence comprising:
a primer sequenced adjacent to, and 5' of, single-stranded
primer sequence a; and
a clamp sequence c1 adjacent to, and 5- of, primer
sequence d, wherein clamp sequence c1 is not complementary to a first template strand
10 sequence i', which is adjacent to, and 3' of, first template strand sequenced';
15
wherein a second strand of the double-stranded primer
sequence comprises primer sequence c1' adjacent to, and 3' of, primer sequence d',
wherein combined sequence cl'-d' is complementary to combined sequence cl-d,
primer sequenced' comprising one or more third modified base(s); and
the first inner primer comprising a single-stranded primer
sequence b that specifically hybridizes to first template strand sequence b', wherein b' is
adjacent to, and 5' of, a', and wherein single-stranded primer sequence b is linked at its
5' end to a first strand of a double-stranded primer sequence comprising:
a primer sequence a adjacent to, and 5' of, single-stranded
20 primer sequence b;
a primer sequenced adjacent to, and 5' of, primer
sequence a; and
a clamp sequence c2 adjacent to, and 5' of, primer
sequence d, wherein clamp sequence c2 is not complementary to first strand template
25 sequence i';
wherein a second strand of the double-stranded primer
sequence comprises primer sequence c2' adjacent to, and 3' of, primer sequenced',
which is adjacent to, and 3' of, primer sequence a', primer sequence a' comprising one
or more fourth modified base(s), wherein combined sequence c2'-d'-a' is
30 complementary to combined sequence c2-d-a;
wherein the unmodified forms of the first and third modified bases
are complementary, and the first and third modified bases preferentially pair with the
unmodified forms, as compared to pairing between the first and third modified bases;
and
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wherein the unmodified forms of the second and fourth modified
bases are complementary, and the second and fourth modified bases preferentially pair
with the unmodified forms, as compared to pairing between the second and fourth
modified bases; and
5 (ii) at least one second primer capable of specifically hybridizing to
the second template strand,
wherein the contacting is carried out under conditions wherein the primers
anneal to their template strands, if present;
(b) amplifying the target nucleic acid, if present, using a DNA polymerase
10 lacking 5' -3' exonuclease activity, under conditions where strand displacement occurs,
to produce amplicons that comprise sequence extending from template sequence a' to the
binding site for the second primer; and
(c) detecting, and optionally quantifying, the target nucleic acid.
12. The primer set or method of any one of claims 9-11, wherein c1 has a different
15 sequence than c2.
20
13. The primer set or method of any one of claims 9-12, wherein the Tm of combined
sequence cl-d, in double-stranded form, is greater than that of combined sequenced-a,
in double-stranded form, and the T m of combined sequence c2-d-a, in double-stranded
form, is greater than that of combined sequence d-a-b, in double-stranded form.
14. The primer set or method of any one of claims 9-13, wherein the second primer
comprises oligonucleotides in the form of, or capable of forming, at least three second
primers capable of hybridizing to the second template strand, wherein the at least three
second primers comprise a second outer primer, a second intermediate primer, and a
second inner primer,
25 the second outer primer comprising a primer sequence h that specifically
hybridizes to second template strand sequence h', primer sequence h comprising one or
more fifth modified base(s);
the second intermediate primer comprising a single-stranded primer sequence e
that specifically hybridizes to second template strand sequence e', wherein e' is adjacent
30 to, and 5' of, h', primer sequence e comprising one or more sixth modified base(s),
wherein single-stranded primer sequence e is linked at its 5' end to a first strand of a
double-stranded primer sequence comprising:
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a primer sequence h adjacent to, and 5' of, single-stranded primer
sequence e; and
a clamp sequence gl adjacent to, and 5' of, primer sequence h, wherein
clamp sequence gl is not complementary to a second template strand sequence j ', which
5 is adjacent to, and 3', of second template strand sequence h';
10
wherein a second strand of the double-stranded primer sequence
comprises primer sequence gl' adjacent to, and 3' of, primer sequence h', wherein
combined sequence gl'-h' is complementary to combined sequence gl-h, primer
sequence h' comprising one or more seventh modified base(s); and
the second inner primer comprising a single-stranded primer sequence f that
specifically hybridizes to first template strand sequence f', wherein f' is adjacent to, and
5' of, e', and wherein single-stranded primer sequence f is linked at its 5' end to a first
strand of a double-stranded primer sequence comprising:
a primer sequence e adjacent to, and 5' of, single-stranded primer
15 sequence f;
a primer sequence h adjacent to, and 5' of, primer sequence e; and
a clamp sequence g2 adjacent to, and 5' of, primer sequence h, wherein
clamp sequence c2 is not complementary to first strand template sequence j ';
wherein a second strand of the double-stranded primer sequence of the
20 inner primer comprises primer sequence g2' adjacent to, and 3' of, primer sequence h',
which is adjacent to, and 3' of, primer sequence e', primer sequence e' comprising one or
more eighth modified base(s), wherein combined sequence g2'-h'-e' is complementary
to combined sequence g2-h-e; and
wherein the unmodified forms of the fifth and seventh modified bases are
25 complementary, and the fifth and sixth modified bases preferentially pair with the
unmodified forms, as compared to pairing between the fifth and seventh modified bases;
and
wherein the unmodified forms of the sixth and eighth modified bases are
complementary, and the sixth and eighth modified bases preferentially pair with the
30 unmodified forms, as compared to pairing between the sixth and eighth modified bases.
15. The primer set or method of claim 14, wherein the Tm of combined sequence glh,
in double-stranded form, is greater than that of combined sequence h-e, in double-
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stranded form, and the T m of combined sequence g2-h-e, in double-stranded form, is
greater than that of combined sequence h-e-f, in double-stranded form.
16. The primer set or method of any one of claims 9-15, wherein clamp sequences c1
and c2, and gl and g2, if present, are not capable of being copied during amplification.
5 17. The primer set or method of claim 16, wherein clamp sequences c1 and c2, and
gland g2, if present, comprise 2'-0-methyl RNA.
18. The primer set or method of any one of the preceding claims, wherein the primer
set comprises, or the method employs, a probe comprising one or more modified bases,
wherein the modified bases preferentially pair with the unmodified bases.
10 19. The primer set or method of any one of the preceding claims, wherein the cycling
probe is a RNase H2 cycling probe.
20. The method of any one of claims 3-8 or 11-19, wherein said amplifying is
carried out in the presence of polyethylene glycol