Real-time polymerase chain reaction-based genotyping assay for single nucleotide polymorphism

Abstract

The present invention provides fluorescence-based real-time PCR assays for the rapid detection of single nucleotide polymorphisms (SNPs). The genotyping assay can be used to detect SNPs of a number of genes of interest that include, but are not limited to, the human multidrug resistance gene (MDR1) single nucleotide polymorphisms C3435T and G2677T, and cytochrome P-450 3A5 single nucleotide polymorphisms CYP3A5*3 (A22893G) and CYP3A5*6 (G30597A).

1 . An isolated DNA molecule useful as a primer for genotyping human multidrug resistance gene (MDR1) single nucleotide polymorphism G2677T, said DNA is selected from the group consisting of SEQ ID NOs: 4-6.  
     
     
         2 . A kit for genotyping human multidrug resistance gene (MDR1) single nucleotide polymorphism G2677T, comprising primers having the DNA sequences of  claim 1 .  
     
     
         3 . A method of genotyping human multidrug resistance gene (MDR1) single nucleotide polymorphism G2677T, comprising the steps of: 
 preparing DNA samples from an individual;    amplifying said DNA with primers SEQ ID NOs: 4 and 6 or SEQ ID NOs: 5 and 6 of  claim 1;  and    identifying the products of said DNA amplification, wherein the presence of products amplified by primers SEQ ID NOs: 5 and 6 indicate said individual has the genotype G2677T.    
     
     
         4 . The method of  claim 3 , wherein said products of DNA amplification are identified by a method selected from the group consisting of real-time fluorescence-based analysis, melt curve analysis and gel electrophoresis.  
     
     
         5 . The method of  claim 4 , wherein said gel electrophoresis identifies a product of 216 base pairs that correspond to genotype G2677T.  
     
     
         6 . An isolated DNA molecule useful as a primer for genotyping human cytochrome P-450 3A5 single nucleotide polymorphism CYP3A5*3, said DNA is selected from the group consisting of SEQ ID NOs: 11-13.  
     
     
         7 . A kit for genotyping human cytochrome P-450 3A5 single nucleotide polymorphism CYP3A5*3, comprising primers having the DNA sequences of  claim 6 .  
     
     
         8 . A method of genotyping human cytochrome P-450 3A5 single nucleotide polymorphism CYP3A5*3, comprising the steps of: 
 preparing DNA samples from an individual; 
 amplifying said DNA with primers SEQ ID NOs: 11 and 13 or SEQ ID NOs: 12 and 13 of  claim 6;  and  
   identifying the products of said DNA amplification, wherein the presence of products amplified by primers SEQ ID NOs: 12 and 13 indicate said individual has the genotype CYP3A5*3.    
     
     
         9 . The method of  claim 8 , wherein said products of DNA amplification are identified by a method selected from the group consisting of real-time fluorescence-based analysis, melt curve analysis and gel electrophoresis.  
     
     
         10 . The method of  claim 9 , wherein said gel electrophoresis identifies a product of 238 base pairs that correspond to genotype CYP3A5*3.  
     
     
         11 . An isolated DNA molecule useful as a primer for genotyping human cytochrome P-450 3A5 single nucleotide polymorphism CYP3A5*6, said DNA is selected from the group consisting of SEQ ID NOs: 14-16.  
     
     
         12 . A kit for genotyping human cytochrome P-450 3A5 single nucleotide polymorphism CYP3A5*6, comprising primers having the DNA sequences of  claim 11 .  
     
     
         13 . A method of genotyping human cytochrome P-450 3A5 single nucleotide polymorphism CYP3A5*6, comprising the steps of: 
 preparing DNA samples from an individual;    amplifying said DNA with primers SEQ ID NOs: 14 and 16 or SEQ ID NOs: 15 and 16 of  claim 11;  and    identifying the products of said DNA amplification, wherein the presence of products amplified by primers SEQ ID NOs: 15 and 16 indicate said individual has the genotype CYP3A5*6.    
     
     
         14 . The method of  claim 13 , wherein said products of DNA amplification are identified by a method selected from the group consisting of real-time fluorescence-based analysis, melt curve analysis and gel electrophoresis.  
     
     
         15 . The method of  claim 14 , wherein said gel electrophoresis identifies a product of 273 base pairs that correspond to genotype CYP3A5*6.