QUANTIFICATION OF MUTANT ALLELES AND COPY NUMBER VARIATION USING DIGITAL PCR WITH NONSPECIFIC DNA-BINDING DYES

摘要

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.

主权项

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, 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.