Polynucleotide Primers and Probes
The present invention provides a novel technology that involves improved primer design. These primer pairs have a wide range of applications and provide high sensitivity and specificity.
1 . A polynucleotide primer combination comprising a first polynucleotide and a second polynucleotide,
the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is complementary to a first target polynucleotide region (T 1 ) and a second domain (Pc) comprising a unique polynucleotide sequence, and
the second polynucleotide (F) comprising a first domain (Fb) having a sequence that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions,
wherein the target polynucleotide has a secondary structure that is denatured by hybridization of Fb to the target polynucleotide.
2 . (canceled)
3 . (canceled)
4 . A polynucleotide primer combination comprising a first polynucleotide and a second polynucleotide,
the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is complementary to a first target polynucleotide region (T 1 ) and a second domain (Pc) comprising a unique polynucleotide sequence, and
the second polynucleotide (F) comprising a first domain (Fb) having a sequence that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions,
wherein P and/or F further comprise a modified nucleic acid.
5 . A polynucleotide primer combination comprising a first polynucleotide, a second polynucleotide, and a blocker polynucleotide,
the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is complementary to a first target polynucleotide region (T 1 ) and a second domain (Pc) comprising a unique polynucleotide sequence,
the second polynucleotide (F) comprising a first domain (Fb) having a sequence that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions, and
the blocker polynucleotide comprising a nucleotide sequence that is complementary to a third target polynucleotide region (T 3 ), wherein T 3 is located 5′ of T 1 and T 2 .
6 . (canceled)
7 . (canceled)
8 . (canceled)
9 . (canceled)
10 . A polynucleotide primer combination comprising a first polynucleotide, a second polynucleotide, and a probe polynucleotide,
the first polynucleotide comprising a first domain (Pa) that is complementary to a first target polynucleotide region (T 1 ) and a second domain (Pc) comprising a unique polynucleotide sequence,
the second polynucleotide (F) comprising a first domain (Fb) that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions, and
the probe polynucleotide comprising a nucleotide sequence that is complementary to a third target polynucleotide region (T 4 ), wherein T 4 is located 5′ of T 1 and T 2 .
11 . (canceled)
12 . (canceled)
13 . (canceled)
14 . (canceled)
15 . A polynucleotide primer combination comprising a first polynucleotide, a second polynucleotide, and a universal quencher polynucleotide,
the first polynucleotide (P) comprising a first domain (Pa) that is complementary to a first target polynucleotide region (T 1 ), a second domain (Pc) comprising a unique polynucleotide sequence, and a label at its 5′ end,
the second polynucleotide (F) comprising a first domain (Fb) that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising two polynucleotide sequences, a 5′ polynucleotide sequence that is sufficiently complementary to the 5′ sequence of Pc such that the 5′ polynucleotide sequence of Pc and Fd will hybridize under appropriate conditions, and a 3′ polynucleotide sequence that is sufficiently complementary to the universal quencher polynucleotide such that the 3′ polynucleotide sequence of Fd and the universal quencher will hybridize under appropriate conditions, and
the universal quencher polynucleotide comprising a quencher and a nucleotide sequence that is sufficiently complementary to the 3′ polynucleotide sequence of Fd such that the universal quencher polynucleotide and the 3′ polynucleotide sequence of Fd will hybridize under appropriate conditions.
16 .- 44 . (canceled)
45 . A method of detecting the presence of a target polynucleotide in a sample with a primer combination, the primer combination comprising a first polynucleotide and a second polynucleotide,
the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is frilly complementary to a first target polynucleotide region (T 1 ) and a second domain (Pc) comprising a unique polynucleotide sequence, Pa having a sequence that is not fully complementary to a non-target polynucleotide in the sample and
the second polynucleotide (F) comprising a first domain (Fb) that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions,
the method comprising the steps of:
contacting the sample with the primer combination and a polymerase under conditions that allow extension of a sequence from Pa which is complementary to the target polynucleotide when the target polynucleotide is present in the sample and
detecting the sequence extended from Pa indicating the presence of the target polynucleotide in the sample.
46 . (canceled)
47 . A method of detecting the presence of a target polynucleotide in a sample with a primer combination of any one of claims 1 to 44 wherein P comprises a first domain that is fully complementary to T 1 and wherein Pa is not fully complementary to a non-target polynucleotide in the sample,
the method comprising the steps of:
contacting the sample with the primer combination and a polymerase under conditions that allow extension of a sequence from Pa which is complementary to the target polynucleotide when the target polynucleotide is present in the sample and
detecting the sequence extended from Pa, wherein detection indicates the presence of the target polynucleotide in the sample.
48 .- 52 . (canceled)
53 . A method of initiating polymerase extension on a target polynucleotide in a sample using a primer combination, the primer combination comprising a first polynucleotide and a second polynucleotide,
the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is fully complementary to a first target polynucleotide region (T 1 ) and a second domain (Pc) comprising a unique polynucleotide sequence, Pa having a sequence that is not fully complementary to a non-target polynucleotide in the sample and
the second polynucleotide (F) comprising a first domain (Fb) that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions,
wherein the sample comprises a mixture of (i) a target polynucleotide that has a sequence (T 1 ) in a first region that is fully complementary to the sequence in Pa and (ii) a non-target polynucleotide that has a sequence (T 1 *) in a first region that is not fully complementary to Pa,
the method comprising the step of contacting the sample with the primer combination and a polymerase under conditions that allow extension of a sequence from Pa and complementary to the target polynucleotide strand when Pa contacts T 1 .
54 . (canceled)
55 . (canceled)
56 . A method of initiating polymerase extension on a target polynucleotide in a sample using a primer combination of any one of claims 1 to 44 , wherein P comprises a first domain (Pa) that is fully complementary to a first target polynucleotide region (T 1 ) and wherein Pa is not fully complementary to a non-target polynucleotide in the sample,
the method comprising the steps of:
contacting the sample with the primer combination and a polymerase under conditions that allow extension of a sequence from Pa which is complementary to the target polynucleotide when the target polynucleotide is present in the sample.
57 .- 61 . (canceled)
62 . A method of amplifying a target polynucleotide in a sample using a polynucleotide primer combination, the primer combination comprising a first polynucleotide and a second polynucleotide,
the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is fully complementary to a first target polynucleotide region (T 1 ) and a second domain (Pc) comprising a unique polynucleotide sequence, Pa having a sequence that is not fully complementary to a non-target polynucleotide in the sample and
the second polynucleotide (F) comprising a first domain (Fb) that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions,
wherein the sample comprises a mixture of (i) a target polynucleotide that has a sequence in a first region (T 1 ) that is fully complementary to the sequence in Pa and (ii) one or more non-target polynucleotides that are not fully complementary to Pa;
the method comprising the steps of:
(a) contacting the sample with the primer combination and a polymerase under conditions that allow extension of a sequence from Pa which is complementary to the target polynucleotide when the target polynucleotide is present in the sample,
(b) denaturing the sequence extended from Pa from the target polynucleotide, and
(c) repeating step (a) in the presence of a reverse primer having a sequence complementary to a region in the sequence extended from Pa in step (b) to amplify the target polynucleotide,
wherein extension and amplification of the target polynucleotide occurs when Pa is fully complementary to the sequence in the Pa but is less efficient or does not occur when the first region in the target polynucleotide is not fully complementary to the sequence in Pa.
63 . A method of amplifying a target polynucleotide in a sample using a polynucleotide primer combination of any one of claims 1 to 44 , wherein the first polynucleotide (P) comprises a first domain (Pa) that is fully complementary to a first target polynucleotide region (T 1 ) and wherein Pa is not fully complementary to a non-target polynucleotide in the sample,
the method comprising the steps of:
(a) contacting the sample with the primer combination and a polymerase under conditions that allow extension of a sequence from Pa which is complementary to the target polynucleotide when the target polynucleotide is present in the sample,
(b) denaturing the sequence extended from Pa from the target polynucleotide, and
(c) repeating step (a) in the presence of a reverse primer having a sequence complementary to a region in the sequence extended from Pa in step (b) to amplify the target polynucleotide,
wherein extension and amplification of the target polynucleotide occurs when T 1 is fully complementary to the sequence in Pa but is less efficient or does not occur when the first region in the target polynucleotide is not fully complementary to the sequence in Pa.
64 . (canceled)
65 . The method of 62 or 63 wherein the reverse primer is a primer combination comprising a first polynucleotide and a second polynucleotide,
the first polynucleotide (PP) comprising a first domain (PPa) having a sequence that is fully complementary to a first region (TT 1 ) in the sequence extended from Pa in step (a) and a second domain (PPc) comprising a unique polynucleotide sequence, and
the second polynucleotide (FF) comprising a first domain (FFb) that is complementary to a second region (TT 2 ) in the sequence extended from Pa in step (a) and a second domain (FFd) comprising a polynucleotide sequence sufficiently complementary to PPc such that PPc and FFd will hybridize under appropriate conditions.
66 . (canceled)
67 . (canceled)
68 . (canceled)
69 . (canceled)