Digital assays for mutation detection

摘要

Provided herein are methods, compositions, and kits for detecting alleles using a single probe or a single primer set. Also, provided herein are methods, compositions, and kits for detecting allelic variants using a single probe or a single primer set. Also, provided herein are methods, compositions, and kits for determining a polymerization error rate.

主权项

1. A method for detecting a first and a second allele of a target locus in a plurality of target polynucleotide molecules, comprising:
(a) partitioning a sample comprising said target polynucleotide molecules into a plurality of fluid reaction volumes; (b) performing an amplification reaction in said reaction volumes, wherein each reaction volume comprises (i) a forward primer that is complementary to a first sequence of a first strand of the target polynucleotide molecules, wherein the first sequence is 5′ of the target locus, (ii) a reverse primer that is complementary to a second sequence of a second strand of the target polynucleotide molecules, wherein the second sequence is 3′ of the target locus, and (iii) a detection probe that is capable of hybridizing to a third sequence of the target polynucleotide molecules that encompasses the target locus, wherein the detection probe comprises a first signal reporter; and (c) determining a number and/or fraction of positive reaction volumes containing a target molecule having the first allele and a number and/or fraction of reaction volumes containing a target molecule having the second allele based on a signal of the first signal reporter detected from the reaction volumes.