Method of detecting a polynucleotide analyte

A method of detecting a single nucleotide polymorphism (SNP) in a polynucleotide analyte in a sample, the method comprising:

• a) contacting the sample comprising the polynucleotide analyte with:
• i) a first nucleic acid probe comprising a 3′ portion complementary to a first portion of the polynucleotide analyte and a 5′ portion that is not complementary to and does not hybridize to the polynucleotide analyte;
• ii) a second nucleic acid probe comprising a 5′ portion complementary to a second portion of said polynucleotide analyte and a 3′ portion that is not complementary to and does not hybridize to the polynucleotide analyte, wherein said first portion of the polynucleotide analyte is 5′ to and contiguous with the second portion of the polynucleotide analyte;
• and
• iii) a structure-specific nucleic acid cleaving agent;
• wherein hybridization of the first nucleic acid probe to the first portion of the polynucleotide analyte and hybridization of the second nucleic acid probe to the second portion of the polynucleotide analyte forms a cleavage structure;
• and
• wherein formation of the cleavage structure allows cleavage of the first nucleic acid probe by the cleaving agent to release a 5′ flap comprising the 5′ portion of the first nucleic acid probe that is not complementary to and does not hybridize to the polynucleotide analyte;
• b) ligating the 5′ flap to a nucleic acid adaptor to form an adaptor-ligated product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3′overhang extension that is complementary to the 5′ flap and wherein the 3′ overhang extension hybridizes to the 5′ flap;
• c) contacting the adaptor-ligated product with:
• i) a type V CRISPR/Cas effector protein;
• ii) a guide RNA comprising a region that binds to the type V CRISPR/Cas effector protein and a guide sequence that is complementary to a portion of the 5′ flap and a portion of the nucleic acid adaptor that is ligated to and adjacent to the 5′ flap;
• and
• iii) a single stranded detector DNA;
• and
• d) measuring a detectable signal produced by cleavage of the single stranded detector DNA by the type V CRISPR/Cas effector protein to detect the adaptor-ligated product, thereby detecting the SNP in the polynucleotide analyte in the sample.

A method of detecting a polynucleotide analyte in a sample, the method comprising

• a) i) contacting the sample comprising the polynucleotide analyte with a first nucleic acid probe and a second nucleic acid probe that are configured to form a cleavage structure in the presence of the polynucleotide analyte;
• and
• ii) a structure-specific nucleic acid cleaving agent;
• wherein formation of the cleavage structure allows cleavage of the first nucleic acid probe by the cleaving agent to release a 5′ flap from the first nucleic acid probe;
• b) ligating the 5′ flap to a nucleic acid adaptor to form an adaptor-ligated product, wherein the nucleic acid adaptor comprises a double-stranded region and a 3′overhang extension that is complementary to the 5′ flap and wherein the 3′ overhang extension hybridizes to the 5′ flap to the nucleic acid adaptor;
• c) contacting the adaptor-ligated product with:
• i) a type V CRISPR/Cas effector protein;
• ii) a guide RNA comprising a region that binds to the type V CRISPR/Cas effector protein and a guide sequence that is complementary to a portion of the 5′ flap and a portion of the nucleic acid adaptor that is ligated to and adjacent to the 5′ flap;
• and
• iii) a single stranded detector DNA;
• and
• d) measuring a detectable signal produced by cleavage of the single stranded detector DNA by the type V CRISPR/Cas effector protein to detect the adaptor-ligated product, thereby detecting the polynucleotide analyte in the sample.