Novel compositions, methods and kits for enhancing pcr specificity

Abstract

The present disclosure provides novel primers and method for the detection of specific nucleic acid sequences. The primers and methods provided herein are useful in a wide variety of molecular biology applications and are particularly useful in allele-specific PCR.

What is claimed is: 
     
         1 . A method of detecting a nucleic acid comprising:
 hybridizing a target nucleic acid with an oligonucleotide comprising a 5′ Sequence-Targeted Amplification Restrictive (STAR) tag sequence and a target nucleic acid hybridization sequence, wherein the STAR tag sequence is capable of forming a stem-loop structure upon extension of the oligonucleotide along the target nucleic acid;   extending the oligonucleotide to form an extension product;   amplifying the extension product to form an amplification product; and   detecting the presence or absence of the amplification product, thereby detecting the target nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein the STAR tag sequence comprises a sequence the same as all or a portion of a target nucleic acid hybridization portion of a second primer to be used with the oligonucleotide in an amplification reaction. 
     
     
         3 . The method of  claim 2 , wherein the STAR tag sequence comprises a sequence the same as all or a portion of a target nucleic acid hybridization portion of the second primer and an internal amplicon sequence of the target nucleic acid. 
     
     
         4 . The method of  claim 1 , wherein the STAR tag sequence is complementary to all or a portion of a second primer to be used with the oligonucleotide in an amplification reaction. 
     
     
         5 . The oligonucleotide of  claim 4 , wherein the STAR tag sequence comprises a sequence complementary to all or a portion of the second primer and an internal amplicon sequence of the target nucleic acid. 
     
     
         6 . The method of  claim 1 , wherein the STAR tag sequence comprises a portion of an internal amplicon sequence of the target nucleic acid. 
     
     
         7 . A method of inhibiting or substantially blocking undesired amplification of a target nucleic acid comprising:
 hybridizing a target nucleic acid with an oligonucleotide comprising a 5′ Sequence-Targeted Amplification Restrictive (STAR) tag sequence and a target nucleic acid hybridization sequence, wherein the STAR tag sequence is capable of forming a stem-loop structure upon extension of the oligonucleotide along the target nucleic acid;   extending the oligonucleotide to form an extension product;   incubating the extension product with an amplification reaction mixture; and   detecting the presence or absence of the extension product.   
     
     
         8 . The method of  claim 7 , wherein the STAR tag sequence comprises a sequence the same as all or a portion of a target nucleic acid hybridization portion of a second primer to be used with the oligonucleotide in an amplification reaction. 
     
     
         9 . The method of  claim 8 , wherein the STAR tag sequence comprises a sequence the same as all or a portion of a target nucleic acid hybridization portion of the second primer and an internal amplicon sequence of the target nucleic acid. 
     
     
         10 . The method of  claim 7 , wherein the STAR tag sequence is complementary to all or a portion of a second primer to be used with the oligonucleotide in an amplification reaction. 
     
     
         11 . The oligonucleotide of  claim 10 , wherein the STAR tag sequence comprises a sequence complementary to all or a portion of the second primer and an internal amplicon sequence of the target nucleic acid. 
     
     
         12 . The method of  claim 7 , wherein the STAR tag sequence comprises a portion of an internal amplicon sequence of the target nucleic acid. 
     
     
         13 . A method of inhibiting or substantially blocking undesired amplification of a target nucleic acid comprising:
 hybridizing a target nucleic acid with an oligonucleotide comprising a 5′ Sequence-Targeted Amplification Restrictive (STAR) tag sequence and a target nucleic acid hybridization sequence, wherein the STAR tag sequence is capable of forming a stem-loop structure upon extension of the oligonucleotide along the target nucleic acid; and   extending the oligonucleotide to form an extension product, wherein the extension product inhibits or substantially reduces undesired amplification of the target nucleic acid.   
     
     
         14 . The method of  claim 13 , wherein the STAR tag sequence comprises a sequence the same as all or a portion of a target nucleic acid hybridization portion of a second primer to be used with the oligonucleotide in an amplification reaction. 
     
     
         15 . The method of  claim 14 , wherein the STAR tag sequence comprises a sequence the same as all or a portion of a target nucleic acid hybridization portion of the second primer and an internal amplicon sequence of the target nucleic acid. 
     
     
         16 . The method of  claim 13 , wherein the STAR tag sequence is complementary to all or a portion of a second primer to be used with the oligonucleotide in an amplification reaction. 
     
     
         17 . The oligonucleotide of  claim 16 , wherein the STAR tag sequence comprises a sequence complementary to all or a portion of the second primer and an internal amplicon sequence of the target nucleic acid. 
     
     
         18 . The method of  claim 13 , wherein the STAR tag sequence comprises a portion of an internal amplicon sequence of the target nucleic acid.