Compositions and methods for detecting rare nucleic acid molecule mutations

A method for detecting and quantifying frequency of a rare nucleic acid molecule deletion mutation, comprising:

• a. contacting a plurality of nucleic acid molecules with a first restriction endonuclease, wherein the first restriction endonuclease is capable of cleaving a nucleic acid molecule comprising a first target region having two or more restriction sites for the first restriction endonuclease, and wherein the nucleic acid molecule is not cleaved by the first restriction endonuclease at the two or more restriction sites of the first target region when the first target region comprises a first deletion mutation;
• b. amplifying the first target region comprising the first deletion mutation from the plurality of nucleic acid molecules of step (a) with a first 5′ primer and a first 3′ primer using digital PCR to obtain a number of the first deletion mutation, wherein the first 5′ primer and the first 3′ primer are complementary to nucleic acid sequences flanking the first target region on the nucleic acid molecule, and only the first target region comprising the first deletion mutation is substantially amplified;
• c. amplifying a control target region from the plurality of nucleic acid molecules of step (a) with a control 5′ primer and a control 3′ primer using digital PCR to obtain a total number of the plurality of nucleic acid molecules, wherein the control 5′ primer and control 3′ primer are complementary to nucleic acid sequences flanking the control target region and the control target region does not comprise a restriction site for the first restriction endonuclease;
• and
• d. obtaining a ratio of the number of the first deletion mutation to the total number of the plurality of nucleic acid molecules;
• thereby detecting and quantifying the frequency of the rare nucleic acid molecule deletion mutation of the first target region within the plurality of nucleic acid molecules.