Methods for variant detection

A method of detecting a variation in a target DNA sequence, the method comprising:

• (a) providing a reaction mixture comprising:
• (i) an oligonucleotide primer having a cleavage domain positioned 5′ of a blocking group, the blocking group linked at or near the end of the 3′-end of the oligonucleotide primer wherein the blocking group prevents primer extension and/or inhibits the primer from serving as a template for DNA synthesis and wherein the oligonucleotide primer contains a 5′ tail sequence that comprises a universal primer sequence, wherein the tail is non-complementary to the target DNA sequence,
• (ii) a sample nucleic acid comprising the target DNA sequence, wherein the target DNA sequence has been altered with a gene editing enzyme, and where the target sequence may or may not have the variation;
• (iii) a hot start cleaving enzyme comprising at least one amino acid substitution as compared to its wild type, wherein the hot start cleaving enzyme is thermostable and has reduced activity at lower temperatures, and wherein the hot start cleaving enzyme exhibits enhanced mismatch discrimination as compared to its wild type;
• and
• (iv) a high-discrimination polymerase;
• (b) hybridizing the oligonucleotide primer to the target DNA sequence to form a double-stranded substrate;
• (c) cleaving the oligonucleotide primer hybridized to the target DNA sequence, if the oligonucleotide primer is complementary at the variation, with the hot start cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the oligonucleotide primer;
• and
• (d) extending the oligonucleotide primer with the high-discrimination polymerase.

A method of target enrichment comprising:

• (a) providing a reaction mixture comprising:
• (i) a first oligonucleotide primer having a tail domain that is not complementary to a target DNA sequence, the tail domain comprising a first universal primer sequence;
• a cleavage domain positioned 5′ of a blocking group, the blocking group linked at or near the end of the 3′-end of the first oligonucleotide primer wherein the blocking group prevents primer extension and/or inhibits the first oligonucleotide primer from serving as a template for DNA synthesis,
• (ii) a sample nucleic acid comprising the target DNA sequence, wherein the target DNA sequence has been altered with a gene editing enzyme;
• (iii) a hot start cleaving enzyme comprising at least one amino acid substitution as compared to its wild type, wherein the hot start cleaving enzyme is thermostable and has reduced activity at lower temperatures, and wherein the hot start cleaving enzyme exhibits enhanced mismatch discrimination as compared to its wild type;
• and
• (iv) a high-discrimination polymerase;
• (b) hybridizing the first oligonucleotide primer to the target DNA sequence to form a double-stranded substrate;
• (c) cleaving the first oligonucleotide primer hybridized to the target DNA sequence, if the first oligonucleotide primer is complementary to the target DNA sequence, with the hot start_cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the first oligonucleotide primer;
• and
• (d) extending the first primer with the high-discrimination polymerase.