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.

1 .- 74 . (canceled) 
     
     
         75 . A method of distinguishing a particular allele in a target nucleic acid molecule, comprising:
 combining said target nucleic acid with a first labeled universal primer, a first allele specific primer, a second labeled universal primer, a second allele specific primer, and a common reverse or forward primer which hybridizes to said target nucleic acid, wherein the particular allele comprises an insertion mutation, a deletion mutation, or a substitution mutation, 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.   
     
     
         76 . The method of  claim 75 , wherein said universal primers are labeled with a single label at or near the 5′ termini. 
     
     
         77 . The method of  claim 75 , wherein said labels are fluorescent. 
     
     
         78 . The method of  claim 77 , wherein at least one of said fluorescent labels is selected from the group consisting of 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE™), fluorescein, N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA™), ALEXA® 450, ALEXA® 594, HEX™, TET™, (5-carboxy-X-rhodamine (5-ROX™), 6-carboxy-X-rhodamine (6-ROX™). 
     
     
         79 . The method of  claim 77 , wherein one of said universal primers is labeled with 5-carboxyfluorescein (FAM), and one of said universal primers is labeled with HEX™. 
     
     
         80 . The method of  claim 79 , wherein the particular allele comprises is a single polynucleotide polymorphism. 
     
     
         81 . A method of detecting an allele in a target nucleic acid, comprising:
 contacting a target nucleic acid with a labeled universal primer, an allele specific primer, and a common reverse or forward primer which hybridizes to said target nucleic acid, wherein the allele comprises an insertion mutation, a deletion mutation, or a substitution mutation, 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.   
     
     
         82 . The method of  claim 81 , wherein the allele comprises an insertion mutation, a deletion mutation, or a substitution mutation. 
     
     
         83 . The method of  claim 81 , wherein the allele comprises a single nucleotide polymorphism. 
     
     
         84 . The method of  claim 81 , wherein said universal primer is labeled at or near the 5′ terminus. 
     
     
         85 . The method of  claim 81 , wherein said label is fluorescent. 
     
     
         86 . The method of  claim 85 , wherein said fluorescent label is selected from the group consisting of 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE™), fluorescein, N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA™), ALEXA® 450, ALEXA® 594, HEX™, TET™, (5-carboxy-X-rhodamine (5-ROX™), 6-carboxy-X-rhodamine (6-ROX™). 
     
     
         87 . The method of  claim 86 , wherein label is 5-carboxyfluorescein (FAM). 
     
     
         88 . The method of  claim 86 , wherein label is HEX™. 
     
     
         89 . The method of  claim 86 , wherein said label is 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE™). 
     
     
         90 . The method of  claim 86 , wherein said label is 6-carboxy-X-rhodamine (ROX).