Quantification by inhibition of amplification

Abstract

The present invention provides a method of quantification of nucleic acid sequences with minimal alterations conducting a real time PCR quantification assay with allele-specific primers. Further, the present invention provides the method for quantifying allelic differences of bovine mitochondrial DNA sequences and allele-specific primers to be used in said method.

What is claimed is:  
     
         1 . A method of quantifying alleles comprising nucleic acid sequence variants coding for the same genetic information, each variant differing in at least one base within a primer-binding site, wherein a variant-specific primer, forming a match with one variant but a mismatch with the other(s) and a probe are added to a real time PCR mixture, resulting in amplification of the one variant that forms a match with said variant-specific primer followed by quantification of said amplified variant by the detection of fluorescence released from the annealed probe during amplification.  
     
     
         2 . The method according to  claim 1 , wherein a non-amplified variant forms a mismatch with a terminal 3′ nucleotide of the variant-specific primer.  
     
     
         3 . The method according to  claim 1 , wherein said variant specific primer comprises additionally at least one mismatch.  
     
     
         4 . The method according to  claim 3 , wherein said additional mismatch is at position −2 to −10 of a 3′ tail of said variant-specific primer.  
     
     
         5 . The method according to  claim 1 , wherein said variant-specific primer forms a match with a minor variant present in smaller amounts than a non-amplified variant(s).  
     
     
         6 . Variant-specific primers with the sequence 
 AS5 (SEQ ID No.: 1)    AS6 (SEQ ID No.: 2)    AS7 (SEQ ID No.: 3)    PIRA1 (SEQ ID No.: 5)    AS1 (SEQ ID No.: 11)    AS2 (SEQ ID No.: 12)    AS3 (SEQ ID No.: 13), or    AS4 (SEQ ID No.: 14).    
     
     
         7 . A method of quantifying alleles comprising variants of mitochondrial nucleic acid sequences, each variant differing in at least one base, wherein a variant-specific primer, forming a match with one variant but a mismatch with the other(s) and a probe are added to a real time PCR mixture, resulting in amplification of the one variant that forms a match with said variant-specific primer followed by quantification of said amplified variant by the detection of fluorescence released from the annealed probe during amplification.  
     
     
         8 . A method of quantifying a nucleic acid sequence variant not quantifiable by a variant-specific primer according to the method of  claim 1 , wherein said variant is quantified by subtracting a copy number of a specifically amplified variant(s) obtained according to the method of  claim 1  from a total copy number of the nucleic acid sequence variants, said total copy number being obtained by a real-time PCR with non-variant-specific primers.  
     
     
         9 . The method of  claim 1 , wherein the variants are mitochondrial nucleic acid sequences, and wherein the variants differ in at least one base.  
     
     
         10 . A method of quantifying alleles comprising variants of mitochondrial nucleic aid sequences, each variant differing in at least one base, wherein a variant-specific primer, forming a match with one variant but a mismatch with the other(s) and a probe are added to a real time PCR mixture, resulting in amplification of the one variant that forms a match with said variant-specific primer followed by quantification of said amplified variant by the detection of fluorescence released from the annealed probe during amplification, and wherein the variant-specific primer is selected from the group consisting of: 
 AS5 (SEQ ID NO:1);    AS6 (SEQ ID NO:2);    AS7 (SEQ ID NO:3);    PIRA1 (SEQ ID NO:5);    ASI (SEQ ID NO:11);    AS2 (SEQ ID NO:12);    AS3 (SEQ ID NO:13); and    AS4 (SEQ ID NO:14).