Methods of Producing and Using Single-Stranded Deoxyribonucleic Acids and Compositions for Use in Practicing the Same

Abstract

Methods of producing single-stranded deoxyribonucleic acids (ssDNAs) are provided. Aspects of the methods include generating a double stranded deoxyribonucleic acid (dsDNA) and then selectively degrading one strand of the dsDNA to produce a ssDNA. ssDNAs produced using methods of the invention find use in a variety of applications, including genomic modification applications. Also provided are compositions, e.g., kits, that find use in practicing various embodiments of the invention.

1 .- 37 . (canceled) 
     
     
         38 . A kit for generating a ssDNA, the kit comprising:
 a nucleic acid comprising a 5′ phosphate or one or more protective base modifications; and   an exonuclease.   
     
     
         39 . The kit according to  claim 38 , wherein the nucleic acid is a ssDNA primer or dsDNA segment comprising a 5′ phosphate. 
     
     
         40 . The kit according to  claim 39 , wherein the dsDNA segment comprises a 5′ phosphate and one or more protective base modifications. 
     
     
         41 . The kit according to  claim 38 , wherein the kit further comprises a ssDNA primer comprising one or more protective base modifications. 
     
     
         42 . The kit according to  claim 38 , wherein the exonuclease is an exonuclease having activity that is blocked by the one or more protective base modifications. 
     
     
         43 . The kit according to  claim 38 , wherein the exonuclease is a 5′-phosphate-dependent exonuclease. 
     
     
         44 . The kit according to  claim 43 , wherein the 5′-phosphate-dependent exonuclease is a lambda exonuclease. 
     
     
         45 . The kit according to  claim 38 , where in the exonuclease is a dsDNA-dependent 3′ to 5′ exonuclease. 
     
     
         46 . The kit according to  claim 45 , wherein the dsDNA-dependent 3′ to 5′ exonuclease is an exonuclease III. 
     
     
         47 . The kit according to  claim 38 , wherein the kit comprises two or more different exonucleases. 
     
     
         48 . The kit according to  claim 47 , wherein the two or more different exonucleases comprise a 5′-phosphate-dependent exonuclease and a dsDNA-dependent 3′ to 5′ exonuclease. 
     
     
         49 . The kit according to  claim 38 , wherein the kit further comprises a PCR polymerase, a ligase or both. 
     
     
         50 . The kit according to  claim 38 , wherein the kit is configured for modifying genomic DNA and further comprises a genome-cleaving nuclease. 
     
     
         51 . The kit according to  claim 50 , wherein the genome-cleaving nuclease is selected from the group consisting of: a Cas9 nuclease, a zinc-finger nuclease and a transcription activator-like effector nuclease. 
     
     
         52 . A kit comprising:
 (a) a double-stranded vector;   (b) a bacterial host cell; and   (c) a cleaving agent.   
     
     
         53 . The kit according to  claim 52 , wherein said double-stranded vector comprises an M13 vector. 
     
     
         54 . The kit according to  claim 52 , wherein said cleaving agent is selected from the group consisting of: a restriction endonuclease and a CRISPR/Cas9 ribonucleoprotein complex. 
     
     
         55 . The kit according to  claim 52 , further comprising instructions for use. 
     
     
         56 . The kit according to  claim 52 , wherein the kit further comprises a PCR polymerase, a ligase or both. 
     
     
         57 . The kit according to  claim 52 , wherein said bacterial host cell is a competent cell.