METHOD OF DESIGNING PRIMERS, METHOD OF DETECTING SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs), METHOD OF DISTINGUISHING SNPs, AND RELATED PRIMERS, DETECTABLE OLIGONUCLEOTIDES, AND KITS

Abstract

A method of designing a primer for detecting a single nucleotide polymorphism (SNP), a method of detecting an SNP, a method of distinguishing SNPs, primers, detectable oligonucleotides, and kits.

What is claimed is: 
     
         1 . A method of detecting at least one mutation (X) of the codon encoding valine at amino acid position 600 (V600X) in exon 15 of the BRAF gene in a sample of nucleic acid from a human, which method comprises:
 (a) performing an amplification reaction with the sample of nucleic acid, wherein the amplification reaction comprises a primer, the last three nucleotides at the 3′ terminus of which encodes X and wherein the fourth nucleotide from the 3′ terminus contains a base other than adenine (A), wherein, if X is present, the primer anneals to X,   wherein, if the sample of nucleic acid is mRNA, step (a) further comprises obtaining cDNA reverse-transcribed from the mRNA or reverse-transcribing cDNA from the mRNA before performing the amplification reaction,   whereupon, if X is present, the amplification reaction produces an amplification product comprising X, and   (b) detecting, and optionally quantitating, the amplification product comprising X,   wherein, if X is encoded by more than one codon, the amplification reaction comprises a primer for each codon,   whereupon a V600X mutation in exon 15 of the BRAF gene in a sample of nucleic acid from a human is detected.   
     
     
         2 . The method of  claim 1 , wherein the amplification reaction further comprises at least one peptide nucleic acid (PNA) clamp, wherein at least one PNA clamp blocks the amplification from wild-type target, and wherein, if the amplification reaction comprises one or more other PNA clamps, the PNA clamps a detectable oligonucleotide and/or a primer by binding an unwanted target and preventing a primer from amplifying from an unwanted target. 
     
     
         3 . The method of  claim 1 , wherein detecting the amplification product comprising X comprises detecting a labeled primer or contacting the amplification product with a detectable oligonucleotide and detecting hybridization of the detectable oligonucleotide to the amplification product comprising X. 
     
     
         4 . The method of  claim 1 , wherein the amplification reaction further comprises an internal control primer, in which case the amplification reaction also produces an amplification product comprising the internal control, in which case step (b) includes detecting the amplification product comprising the internal control. 
     
     
         5 . The method of  claim 4 , wherein detecting the amplification product comprising the internal control comprises detecting a labeled primer or contacting the amplification product with a detectable oligonucleotide and detecting hybridization of the detectable oligonucleotide to the amplification product comprising the internal control. 
     
     
         6 . The method of  claim 1 , wherein X is at least one amino acid selected from the group consisting of E, K, D R, and N. 
     
     
         7 . The method of  claim 1 , wherein X is at least one amino acid selected from the group consisting of E, K, and D. 
     
     
         8 . The method of  claim 1 , wherein X is E. 
     
     
         9 . The method of  claim 1 , wherein X is E and K, E and D, or K and D. 
     
     
         10 . The method of  claim 1 , wherein X is E, K, and D. 
     
     
         11 . The method of  claim 1 , wherein, when the method comprises detecting two or more X, the method can comprise performing an amplification reaction with the sample of DNA for each X together or separately. 
     
     
         12 . The method of  claim 11 , wherein the method further comprises determining which X is present in the sample of DNA. 
     
     
         13 - 25 . (canceled) 
     
     
         26 . A method of detecting at least one mutation (X) of a codon in a gene in a sample of nucleic acid, which method comprises:
 (a) performing an amplification reaction with the sample of nucleic acid, wherein the amplification reaction comprises a primer, the last three nucleotides at the 3′ terminus of which encodes X and wherein the fourth nucleotide from the 3′ terminus contains a base other than adenine (A), wherein, if X is present, the primer anneals to X,   wherein, if the sample of nucleic acid is mRNA, step (a) further comprises obtaining cDNA reverse-transcribed from the mRNA or reverse-transcribing cDNA from the mRNA before performing the amplification reaction,   whereupon, if X is present, the amplification reaction produces an amplification product comprising X,   (b) detecting the amplification product comprising X, and   wherein, if X is encoded by more than one codon, the amplification reaction comprises a primer for each codon,   whereupon a mutation is detected in the codon of the gene in a sample of nucleic acid from a human is detected.   
     
     
         27 . The method of  claim 26 , wherein the amplification reaction further comprises at least one PNA clamp, wherein at least one PNA clamp blocks the amplification from wild-type target, and wherein, if the amplification reaction comprises one or more other PNA clamps, the PNA clamps a detectable oligonucleotide and/or a primer by binding an unwanted target and preventing a primer from amplifying from an unwanted target. the PNA clamps a detectable oligonucleotide and/or a primer. 
     
     
         28 . The method of  claim 26 , wherein detecting the amplification product comprising X comprises detecting a labeled primer or contacting the amplification product with a detectable oligonucleotide and detecting hybridization of the detectable oligonucleotide to the amplification product comprising X. 
     
     
         29 . The method of  claim 26 , wherein the amplification reaction further comprises an internal control primer, in which case the amplification reaction also produces an amplification product comprising the internal control, in which case step (b) includes detecting the amplification product comprising the internal control. 
     
     
         30 . The method of  claim 29 , wherein detecting the amplification product comprising the internal control comprises detecting a labeled primer or contacting the amplification product with a detectable oligonucleotide and detecting hybridization of the detectable oligonucleotide to the amplification product comprising the internal control. 
     
     
         31 . The method of  claim 29 , wherein, when the method comprises detecting two or more X, the method can comprise performing an amplification reaction with the sample of DNA for each X together or separately. 
     
     
         32 . A method of designing a primer for detection of at least one mutation (X) of a codon in a gene in a sample of nucleic acid, which method comprises synthesizing a primer, the last three nucleotides at the 3′ terminus of which encodes X and wherein the fourth nucleotide from the 3′ terminus contains a base other than that which is present in the wild-type gene, whereupon a primer for detection of at least one mutation (X) in a codon in a gene in a sample of nucleic acid is designed.