Patent ID: 9322006
Filing Date: 2016-04-26
CPC Classification: A61P,C12N,C12Q

Claim Text:
1. A method comprising (a) providing a dimeric nuclease that cuts one or more double-stranded target sites of a double-stranded nucleic acid and creates a 5′ overhang on the double-stranded nucleic acid, wherein the dimeric nuclease comprises two nuclease monomers, and wherein each of the target sites comprises (i) a left-half site, wherein the left-half site comprises a nucleic acid sequence that is bound by one monomer of the dimeric nuclease; (ii) a right-half site, wherein the right-half site comprises a nucleic acid sequence that is bound by other monomer of the dimeric nuclease; and (iii) a spacer sequence between the left-half site and the right-half site; wherein the left-half site, the spacer sequence, and right-half site forms a 5′-[the left-half site]-[the spacer sequence]-[the right-half site]-3′ (LSR) structure, and the cleavage site of the dimeric nuclease is located within the spacer sequence; (b) contacting the dimeric nuclease with a library of candidate nucleic acid molecules, wherein each of the candidate nucleic acid molecules comprises a concatemer containing multiple copies of identical DNA sequences, a plurality of the target sites and multiple constant insert sequences, each of the constant insert sequences is located between two target sites of the plurality of the target sites, and each of the multiple copies of the identical DNA sequences comprises a target site of the plurality of the target sites and a constant insert sequence of the multiple constant insert sequences, under conditions suitable for the dimeric nuclease to cut a candidate nucleic acid molecule of the library of candidate nucleic acid molecules; thereby generating one or more candidate nucleic acid molecules cut once, twice and multiple times by the dimeric nuclease, wherein the candidate nucleic acid molecules cut twice by the dimeric nuclease comprise a 5′ overhang and the constant insert sequence flanked by the left half-site and a part of the spacer sequence from one of the plurality of the target sites, and flanked by the right half-site and a part of the spacer sequence from another of the plurality of the target sites, (c) filling in the 5′ overhang of each of the one or more candidate nucleic acid molecules cut twice by the dimeric nuclease, thereby creating one or more candidate nucleic acid molecules with blunt ends; and (d) identifying the one or more target sites of the one or more candidate nucleic acid molecules cut twice by the dimeric nuclease by determining the sequence of the one or more candidate nucleic acid molecules with blunt ends created in step (c).