Compositions for improved allele-specific pcr

Abstract

The present invention includes compositions for allele-specific amplification, utilizing an allele-specific oligonucleotide, at least partially complementary to more than one variant of the target sequence, but having at least one selective nucleotide complementary to only one variant of the target sequence and incorporating at least one G-damp nucleotide.

1 . A kit for allele-specific amplification of a target sequence, said target existing in the form of several variant sequences, the kit comprising:
 (a) a first oligonucleotide, comprising a sequence at least partially complementary to a portion of one or more variant of the target sequence; and   (b) a second oligonucleotide, comprising a sequence at least partially complementary to a portion of one or more variants of the target sequence, and further comprising one selective nucleotide at the 3′ terminal nucleotide that is complementary to only one variant of the target sequence, and at least one G-clamp nucleotide at a position between 1 and 5 nucleotides upstream of the 3′ terminal nucleotide.   
     
     
         2 . The kit of  claim 1 , further comprising a nucleic acid polymerase, nucleoside triphosphates, buffer suitable for the extension of nucleic acids by the nucleic acid polymerase and a set of instructions for performing allele-specific amplification. 
     
     
         3 . The kit of  claim 2 , wherein the nucleic acid polymerase is selected from a group consisting of Taq DNA polymerase, Z05 DNA polymerase, ΔZ05 DNA polymerase and ΔZ05-Gold DNA polymerase. 
     
     
         4 . The kit of  claim 2 , wherein the nucleic acid polymerase possesses 3′-5′ nuclease activity. 
     
     
         5 . The kit of  claim 4 , wherein the nucleic acid polymerase is selected from a group consisting of Pfu DNA polymerase and  Thermatoga Maritima  polymerases. 
     
     
         6 . The kit of  claim 1 , wherein the target sequence is the EGFR gene. 
     
     
         7 . The kit of  claim 1 , wherein the second oligonucleotide has a sequence selected from a group consisting of SEQ ID NO: 6, 7 and 12. 
     
     
         8 . A reaction mixture for allele-specific amplification of a target sequence, said target existing in the form of several variant sequences, the reaction mixture comprising:
 (a) a first oligonucleotide, comprising a sequence at least partially complementary to a portion of one or more variant of the target sequence; and   (b) a second oligonucleotide, comprising a sequence at least partially complementary to a portion of one or more variants of the target sequence, and further comprising one selective nucleotide at the 3′ terminal nucleotide that is complementary to only one variant of the target sequence, and at least one G-clamp nucleotide at a position between 1 and 5 nucleotides upstream of the 3′ terminal nucleotide.   
     
     
         9 . The reaction mixture of  claim 8 , further comprising a nucleic acid polymerase, nucleoside triphosphates and a buffer suitable for the extension of nucleic acids by the nucleic acid polymerase. 
     
     
         10 . The reaction mixture of  claim 9 , wherein the nucleic acid polymerase is selected from a group consisting of Taq DNA polymerase, Z05 DNA polymerase, ΔZ05 DNA polymerase and ΔZ05-Gold DNA polymerase. 
     
     
         11 . The reaction mixture of  claim 9 , wherein the nucleic acid polymerase possesses 3′-5′ nuclease activity. 
     
     
         12 . The reaction mixture of  claim 11 , wherein the nucleic acid polymerase is selected from a group consisting of Pfu DNA polymerase and  Thermatoga Maritima  polymerases. 
     
     
         13 . The reaction mixture of  claim 8 , wherein the target sequence is the EGFR gene. 
     
     
         14 . The reaction mixture of  claim 8 , wherein the second oligonucleotide has a sequence selected from a group consisting of SEQ ID NO: 6, 7 and 12.