Methods and kits for targeted cleavage and enrichment of Nucleic Acids for high-throughput analyses of user-defined genomic regions

A method for enriching a sequence of interest from a source sequence for sequencing comprising:

• I. obtaining a source DNA,
• II. denaturing the source DNA,
• III. annealing a pair of flap oligonucleotides 1-N and 2-N to a first and second region, respectively, of the denatured source sequence, each flap oligonucleotide 1-N and 2-N hybridized to a universal oligonucleotide 1-N(U1-N), wherein the first region comprises a portion of the sequence of interest and wherein the second region is downstream of the sequence of interest,
• IV. cleaving the denatured source DNA using a structure-specific endonuclease to produce a cleaved sequence of interest comprising 5′ and 3′ cut ends,
• V. ligating the 5′ cut end to the oligonucleotide U1-N such that the sequence of interest comprises a ligated U1-N hybridized with the flap oligonucleotide 1-N,
• VI. ligating the 3′ cut end to an oligonucleotide U2 hybridized to an oligonucleotide 3-N, wherein a 5′ portion of the oligonucleotide 3-N is sufficiently complementary to U2 and a 3′ tail portion is sufficiently complementary to a portion of the sequence of interest such that the sequence of interest comprises a ligated oligonucleotide U2 hybridized to oligonucleotide 3-N, and
• VII. subjecting the sequence of interest from step (V), step (VI), or both, to one or more exonucleases to produce an enriched single strand of DNA comprising an arrangement of U1-N-Sequence of interest-U2.

A method for enriching a sequence of interest from a source sequence comprising:

• I. obtaining a source DNA,
• II. denaturing the source DNA,
• III. annealing a pair of flap oligonucleotides 1-N and oligonucleotide 2-N to a first and second region, respectively, of the denatured source sequence, each flap oligonucleotides 1-N and 2-N hybridized to a universal oligonucleotide-1 (U1), wherein the first region comprises a portion of the sequence of interest and wherein the second region is downstream of the sequence of interest,
• IV. cleaving the denatured source DNA using a structure-specific endonuclease to produce a cleaved sequence of interest comprising 5′ and 3′ cut ends,
• V. ligating the 3′ cut end to an oligonucleotide oligo U2 hybridized to an oligonucleotide 3-N, wherein a 5′ portion of the oligonucleotide 3-N is complementary to oligo U2 and a 3′ tail portion is complementary to a portion of the sequence of interest such that the sequence of interest comprises a ligated oligonucleotide U2 hybridized to oligonucleotide 3,
• VI. subjecting the sequence of interest from step (V), to one or more exonucleases to produce a single strand of DNA comprising an arrangement of Sequence of interest-U2,
• VII. annealing to the enriched sequence of interest-U2 to a flap oligonucleotide 1-N hybridized to a universal oligonucleotide 1-N(U1-N),
• VIII. ligating oligonucleotide U1-N to the 5′ end of the sequence of interest-U2,
• IX. subjecting the sequence of interest-U2 of step VIII to one or more exonucleases to produce a single strand of DNA comprising an arrangement of U1-N-Sequence of interest-U2.