Detection of single nucleotide polymorphisms in human kras

Abstract

Disclosed herein are primers and probes for the detection of single nucleotide polymorphisms (SNPs) in the KRAS gene. These primers and probes may be used in a method of identifying the presence or absence of one or more SNPs in the KRAS gene. These primers and probes may also be used in a method of predicting a response of a subject in need thereof to a cancer therapy. The primers and probes may further be used in a method of selecting a cancer therapy for a subject in need thereof.

1 .- 18 . (canceled) 
     
     
         19 . A method of identifying the presence or absence of one or more single nucleotide polymorphisms (SNPs) in v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), the method comprising:
 (a) forming a reaction mixture comprising
 (i) a sample obtained from the subject, wherein the sample is suspected of containing a KRAS target sequence and 
 (ii) a primer and probe set comprising:
 A. one or more forward primer nucleic acid sequences comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9; 
 B. one or more reverse primer nucleic acid sequences comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; 
 C. one or more probe nucleic acid sequences comprising SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42; and 
 D. one or more peptide nucleic oligomer sequences comprising SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57; 
 
   (b) subjecting the reaction mixture to conditions sufficient for formation of one or more amplicons; and   (c) detecting the one or more amplicons.   
     
     
         20 . The method of  claim 19 , wherein the reaction mixture further comprises an internal control comprising:
 (iii) a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45;   (iv) a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48; and   (v) a probe comprising a nucleotide sequence as set forth in SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51.   
     
     
         21 . The method of  claim 20 , further comprising determining a difference in cycle number (ΔCN), wherein the ΔCN is a difference between a cycle number associated with an amplicon containing the SNP and a cycle number associated with an amplicon generated from the internal control. 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 19 , further comprising identifying the presence of one or more SNPs in KRAS based upon the detection in step (c). 
     
     
         24 . The method of  claim 23 , wherein the one or more SNPs are selected from G12C, G12R, G12S, G12D, G12A, G12V, G13D, G13C, and Q61H. 
     
     
         25 . The method of  claim 19 , wherein the one or more probe nucleic acid sequences comprises a detectable label. 
     
     
         26 . The method of  claim 25 , wherein the detectable label is a fluorescent label. 
     
     
         27 .- 28 . (canceled) 
     
     
         29 . The method of  claim 26 , wherein detecting includes measuring a fluorescent signal generated by the detectable label. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 19 , wherein the sample comprises cancerous tissue, a blood sample, a serum sample, a plasma sample, or circulating tumor DNA (ctDNA). 
     
     
         32 .- 58 . (canceled)