Methods and compositions for recombinase-mediated selective cleavage of nucleic acids

Abstract

Embodiments of the methods and compositions provided herein relate to the selective cleavage of target nucleic acids. Some embodiments include recombinase-mediated selective cleavage of target nucleic acids with single-stranded nucleic acid probes and a recombinase. Some embodiments also include the enrichment of non-target nucleic acids in a sample by selective cleavage of target nucleic acids in the sample, and removal of the cleaved target nucleic acids from the sample.

What is claimed is: 
     
         1 - 70 . (canceled) 
     
     
         71 . A kit for selectively cleaving a target nucleic acid in a sample, the kit comprising:
 (i) reagents for forming a D-loop in a double-stranded target nucleic acid comprising a recombinase and a single-stranded nucleic acid probe capable of hybridizing to the target nucleic acid; and   (ii) a first nuclease for cleaving a target nucleic acid at a D-loop.   
     
     
         72 . The kit of  claim 71 , further comprising a polymerase and a plurality of nucleotides. 
     
     
         73 . The kit of  claim 72 , further comprising a second nuclease for degrading an extended single-stranded nucleic acid probe. 
     
     
         74 . The kit of  claim 73 , wherein the plurality of nucleotides comprise dUTP nucleotides. 
     
     
         75 . The kit of  claim 74 , wherein the second nuclease is selected from the group consisting of uracil DNA glycosylase, apurinic/apyrimidinic endonuclease, and DNA glycosylase endonuclease VII. 
     
     
         76 . The kit of  claim 72 , wherein the plurality of nucleotides comprise a terminator nucleotide. 
     
     
         77 . The kit of  claim 71 , further comprising a single-stranded binding protein. 
     
     
         78 . The kit of  claim 71 , wherein the recombinase is selected from the group consisting of: RecA, UvsX, RAD51, and derivatives thereof. 
     
     
         79 . The kit of  claim 78 , wherein the recombinase comprises an UvsX recombinase. 
     
     
         80 . The kit of  claim 71 , wherein the first nuclease is a single-stranded specific endonuclease. 
     
     
         81 . The kit of  claim 80 , wherein the first nuclease is selected from a S1 nuclease and a mung bean nuclease. 
     
     
         82 . The kit of  claim 71 , wherein the first nuclease comprises a recombinant protein comprising a nuclease domain and single-stranded nucleic acid binding domain. 
     
     
         83 . The kit of  claim 82 , wherein the nuclease domain is derived from a FokI protein or a TevI protein, and the single-strand binding domain is derived from a Sso7d protein. 
     
     
         84 . The kit of  claim 71 , wherein the single-stranded nucleic acid probe has a length in a range from 10 nucleotides to 100 nucleotides. 
     
     
         85 . The kit of  claim 84 , wherein the single-stranded nucleic acid probe has a length in a range from 30 nucleotides to 60 nucleotides. 
     
     
         86 . The kit of  claim 71 , wherein the single-stranded nucleic acid probe is complementary to a sequence selected from the group consisting of mitochondrial DNA, and a repetitive element selected from the group consisting of an interspersed repeat, a tandem repeat, and a long terminal repeat. 
     
     
         87 . The kit of  claim 71 , wherein the single-stranded nucleic acid probe is obtained from a fragment of genomic DNA. 
     
     
         88 . The kit of  claim 71 , wherein the single-stranded nucleic acid probe is obtained from a cDNA. 
     
     
         89 . The kit of  claim 71 , further comprising a plurality of single-stranded nucleic acid probes. 
     
     
         90 . The kit of  claim 89 , wherein the plurality of single-stranded nucleic acid probes comprises different single-stranded nucleic acid probes.