Method for Removing and/or Detecting Nucleic Acids Having Mismatched Nucleotides

Abstract

Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may comprise ligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.

1 - 10 . (canceled) 
     
     
         10 . A kit comprising:
 (a) a T-tailed double-stranded adapter; and   (a) an EndoMS enzyme.   
     
     
         11 . The kit of  claim 10 , wherein the kit further comprises a ligase. 
     
     
         12 . The kit of  claim 10 , wherein the kit further comprises a reaction buffer. 
     
     
         13 . A method, comprising:
 (a) hybridizing a first single stranded nucleic acid with a second single stranded nucleic acid that is not perfectly complementary to a target sequence in the first nucleic acid to produce a duplex nucleic acid that comprises one or more mismatches; and   (b) treating the duplex nucleic acid with EndoMS, so as to cleave the duplex nucleic acid at the target sequence.   
     
     
         14 . The method of  claim 13 , wherein the second single stranded nucleic acid is an oligonucleotide. 
     
     
         15 . The method of  claim 13 , wherein the first single stranded nucleic acid is denatured dsDNA. 
     
     
         16 . The method of  claim 13 , wherein the hybridizing is done by strand invasion. 
     
     
         17 . The method of  claim 13 , wherein, in the duplex, the first and second nucleic acids comprise at least 8 nucleotides of perfectly complementarity on each side of a mismatch. 
     
     
         18 . A method for identifying a mismatched nucleotide in a double-stranded nucleic acid, comprising:
 (a) reacting a sample comprising the double-stranded nucleic acid with EndoMS to produce a reaction product, wherein the EndoMS cleaves both strands of the double-stranded nucleic acid only if it contains a mismatch;   (b) subjecting the reaction product of (a) to amplification under conditions that amplify the double-stranded nucleic acid if it is uncleaved but not if it is cleaved; and   (c) detecting the presence of an amplification product, wherein the presence of the product indicates that the double-stranded nucleic acid does not have a mismatched nucleotide and the absence of a product indicates that the double-stranded nucleic acid has a mismatched nucleotide.   
     
     
         19 . The method of  claim 18 , wherein step (b) is done by PCR, using primers that flank the mismatch. 
     
     
         20 . The method of  claim 18 , wherein mismatch is at a ligation junction. 
     
     
         21 . The method of  claim 18 , wherein the double-stranded nucleic acid is an adapter dimer. 
     
     
         22 . The method of  claim 18 , wherein the double-stranded nucleic acid is genomic DNA. 
     
     
         23 . The method of  claim 18 , wherein the detecting step (c) comprises quantifying the amount of the amplification product. 
     
     
         24 . A method of cleaving a double-stranded adapter dimer, the method comprising incubating the adapter dimer with EndoMS to form adapter dimer cleavage products, wherein the adapter dimer comprises at least one mismatched oligonucleotide. 
     
     
         25 . A method according to  claim 24 , wherein the cleavage products comprise products corresponding to cleavage of both strands of the adapter dimer. 
     
     
         26 . A method according to  claim 24 , wherein the adaptor dimer is a loop adapter or a hairpin adaptor. 
     
     
         27 . A method of cleaving a double-stranded nucleic acid molecule, the method comprising incubating the double-stranded nucleic acid molecule with EndoMS to form cleavage products, wherein the double-stranded nucleic acid molecule comprises at least one mismatched oligonucleotide. 
     
     
         28 . A method according to  claim 27 , wherein the cleavage products comprise products corresponding to cleavage of both strands of the double-stranded nucleic acid molecule. 
     
     
         29 . A method according to  claim 27 , wherein the double-stranded nucleic acid molecule is an adapter dimer. 
     
     
         30 . A method according to  claim 27 , wherein the adaptor dimer is a loop adapter or a hairpin adaptor.