Compositions and methods for the detection and discrimination of nucleic acids
First Claim
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1. A method of distinguishing a single nucleotide polymorphism (SNP) in a target nucleic acid, comprising:
- combining said target nucleic acid with a first internally labeled universal primer, a first allele specific primer, a second internally labeled universal primer, a second allele specific primer, a reverse or forward primer which hybridizes to said target nucleic acid, and a nonspecific double stranded DNA binding protein, wherein said first and second labels are different, and wherein said first allele specific primer has a 5′
tail which hybridizes to said first universal primer and a terminal 3′
nucleotide that is specific to a first allele, and said second allele specific primer has a 5′
tail which hybridizes to said second universal primer, and a terminal 3′
nucleotide that is specific to a second allele, under conditions which allow extension of said allele specific primers; and
detecting said first and said second labels, wherein the presence of said first label indicates the presence of said first allele, the presence of said second label indicates the presence of said second allele, and the presence of both labels indicates the presence of both alleles.
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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.
121 Citations
19 Claims
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1. A method of distinguishing a single nucleotide polymorphism (SNP) in a target nucleic acid, comprising:
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combining said target nucleic acid with a first internally labeled universal primer, a first allele specific primer, a second internally labeled universal primer, a second allele specific primer, a reverse or forward primer which hybridizes to said target nucleic acid, and a nonspecific double stranded DNA binding protein, wherein said first and second labels are different, and wherein said first allele specific primer has a 5′
tail which hybridizes to said first universal primer and a terminal 3′
nucleotide that is specific to a first allele, and said second allele specific primer has a 5′
tail which hybridizes to said second universal primer, and a terminal 3′
nucleotide that is specific to a second allele, under conditions which allow extension of said allele specific primers; and
detecting said first and said second labels, wherein the presence of said first label indicates the presence of said first allele, the presence of said second label indicates the presence of said second allele, and the presence of both labels indicates the presence of both alleles. - View Dependent Claims (2, 3, 4)
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5. A method of detecting an allele in a target nucleic acid, comprising:
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Contacting a target nucleic acid with an internally labeled universal primer, an allele specific primer, a common reverse or forward primer which hybridizes to said target nucleic acid, and a nonspecific single stranded DNA binding protein, wherein said allele specific primer has a 5′
tail which hybridizes to said universal primer and a terminal 3′
nucleotide that is specific to said allele, under conditions which allow extension of said allele specific primer; and
Detecting said label, wherein the presence of said label indicates the presence of said allele. - View Dependent Claims (6, 7, 8, 9)
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- 10. A composition comprising a first internally labeled universal primer and a nonspecific double stranded DNA binding protein.
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14. A composition comprising a first internally labeled universal primer, a first allele specific primer, a second internally labeled universal primer, a second allele specific primer, a common reverse or forward primer which hybridizes to a target nucleic acid, and a nonspecific double stranded DNA binding protein, wherein said first and second labels are different, and wherein said first allele specific primer has a 5′
- tail which hybridizes to said first universal primer and a terminal 3′
nucleotide that is specific to a first allele, and said second allele specific primer has a 5′
tail which hybridizes to said second universal primer, and a terminal 3′
nucleotide that is specific to said second allele - View Dependent Claims (15)
- tail which hybridizes to said first universal primer and a terminal 3′
- 16. A method of reducing the fluorescence of a fluorescently labeled hairpin primer, comprising contacting said primer with a nonspecific double stranded DNA binding protein.
Specification