Primers 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, and a common reverse or forward primer which hybridizes to said target nucleic acid, wherein said first and second internally labeled universal primers are labeled with a single label that undergoes a detectable change in one or more detectable properties upon hybridization or extension, 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.
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Citations
8 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, and a common reverse or forward primer which hybridizes to said target nucleic acid, wherein said first and second internally labeled universal primers are labeled with a single label that undergoes a detectable change in one or more detectable properties upon hybridization or extension, 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; anddetecting 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)
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4. 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, and a common reverse or forward primer which hybridizes to said target nucleic acid, wherein said internally labeled universal primer is labeled with a single label that undergoes a detectable change in one or more detectable properties upon hybridization or extension, 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; anddetecting said label, wherein the presence of said label indicates the presence of said allele. - View Dependent Claims (5, 6, 7, 8)
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Specification
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Current AssigneeLife Technologies Corporation (Thermo Fisher Scientific Incorporated)
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Original AssigneeLife Technologies Corporation (Thermo Fisher Scientific Incorporated)
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InventorsNazarenko, Irina, Rashtchian, Ayoub, Solus, Joseph, Darfler, Marlene, Gebeyehu, Gulilat, Astatke, Mekbib, Pires, Richard
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Primary Examiner(s)Strzelecka, Teresa E
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Assistant Examiner(s)Pande, Suchira
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Application NumberUS12/629,045Publication NumberTime in Patent Office1,197 DaysField of SearchNoneUS Class Current435/91.2CPC Class CodesC12P 19/34 Polynucleotides, e.g. nucle...C12Q 1/6816 characterised by the detect...C12Q 1/6827 for detection of mutation o...C12Q 1/686 Polymerase chain reaction [...C12Q 1/6869 Methods for sequencingC12Q 1/6876 Nucleic acid products used ...C12Q 2525/121 incorporating both deoxyrib...C12Q 2525/161 incorporating target specif...C12Q 2563/107 fluorescenceC12Q 2565/107 Alteration in the property ...G01N 21/6486 Measuring fluorescence of b...
