Quantification of Mutant Alleles and Copy Number Variation Using Digital PCR with Nonspecific DNA-Binding Dyes

Abstract

Methods and reagents for performing digital PCR for detection and quantification of mutant alleles and copy number variation are disclosed. In particular, the invention relates to methods using a nonspecific DNA-binding dye, which produces a fluorescent signal that increases in intensity according to the number of base-pairs present in the PCR amplicon product. The method utilizes mutant-specific and wild-type-specific primers having non-complementary “tail” sequences of different lengths. Accordingly, the amplicons for the wild-type and mutant alleles differ in length and can be distinguished based on the difference in the intensities of their fluorescent signals. The methods of the invention can be used to detect rare genetic events, including single nucleotide mutations, alterations of copy number, and deletions or insertions of nucleotides.

What is claimed is: 
     
         1 . A composition for performing digital polymerase chain reaction (PCR), the composition comprising:
 a) a first set of primers for amplification of a region of interest in a target polynucleotide sequence, wherein each primer comprises a 5′-tail of a first length;   b) a second set of primers for amplification of a reference polynucleotide sequence, wherein each primer comprises a 5′-tail of a second length, wherein the shorter of the two 5′ tails has a higher GC content, and the longer of the 5′ tails has a higher AT content, such that amplifying nucleic acids with said primers will result in amplicons of the region of interest and amplicons of the reference polynucleotide sequence having different lengths; and   c) a fluorescent DNA dye.   
     
     
         2 . The composition of  claim 1 , wherein the fluorescent DNA dye is selected from the group consisting of EvaGreen (EG), SYBR green, SYBR green II, SYBR gold, Oxazole yellow (YO), YOYO, Thiazole orange (TO), PicoGreen (PG), and a SYTO dye. 
     
     
         3 . The composition of  claim 1 , wherein the fluorescent DNA dye is EvaGreen (EG) dye. 
     
     
         4 . The composition of  claim 1 , further comprising reagents for performing droplet digital PCR. 
     
     
         5 . The composition of  claim 1 , wherein the region of interest comprises a mutation and the reference polynucleotide sequence comprises a wild-type sequence. 
     
     
         6 . The composition of  claim 5 , wherein the mutation is a single nucleotide variation. 
     
     
         7 . The composition of  claim 5 , wherein the mutation is in a target polynucleotide sequence from a genetically aberrant cell, rare blood cell, or cancerous cell. 
     
     
         8 . The composition of  claim 1 , wherein each set of primers is designed to produce amplicons up to 500 base pairs in length. 
     
     
         9 . The composition of  claim 1 , wherein a melting temperature for primer-template hybridization complexes for the target and reference polynucleotides are matched. 
     
     
         10 . The composition of  claim 1 , wherein a melting temperature for primer-template hybridization complexes for the target and reference polynucleotides are within 3° C. of each other. 
     
     
         11 . The composition of  claim 1 , wherein the primers have a length of between 20 and 40 nucleotides. 
     
     
         12 . A kit for performing digital PCR, the kit comprising:
 a) written instructions for performing digital PCR;   b) a first set of primers for amplification of a region of interest in a target polynucleotide sequence, wherein each primer comprises a 5′-tail of a first length;   c) a second set of primers for amplification of a reference polynucleotide sequence, wherein each primer comprises a 5′-tail of a second length, wherein the shorter of the two 5′ tails has a higher GC content, and the longer of the 5′ tails has a higher AT content, such that amplifying nucleic acids with said primers will result in amplicons of the region of interest and amplicons of the reference polynucleotide sequence having different lengths; and   d) a fluorescent DNA dye.   
     
     
         13 . The kit of  claim 12 , further comprising a polymerase and buffers. 
     
     
         14 . The kit of  claim 12 , further comprising reagents for performing droplet digital PCR.