Methods for Fragmentation and Labeling of Nucleic Acids

Abstract

The invention provides methods, compositions, and kits for fragmentation and labeling of nucleic acids. More particularly, the invention relates to methods for fragmentation of nucleic acids to produce fragments with 3′ end hydroxyl groups within a desired size range. In methods of the invention, nucleic acids are fragmented at abasic sites to produce fragments with blocked 3′ ends. The 3′ ends are unblocked to produce polynucleotide fragments with hydroxyl groups at their 3′ ends. Methods, kits, and compositions for carrying out fragmentation of a polynucleotide template in a single reaction mixture to yield fragments with 3′-hydroxl ends within the desired size range are disclosed.

1 - 74 . (canceled) 
     
     
         75 . A kit comprising:
 (a) an agent capable of cleaving a base portion of at least one non-canonical nucleotide from a polynucleotide, wherein the polynucleotide comprises at least one non-canonical nucleotide, thereby generating an abasic site in the polynucleotide;   (b) an agent capable of fragmenting the phosphodiester backbone of the polynucleotide at or near the abasic site, thereby forming a polynucleotide fragment with a blocked 3′ end;   (c) an enzyme capable of unblocking the blocked 3′ end of the polynucleotide fragment, wherein the enzyme comprises a nonprocessive 3′ to 5′ exonuclease activity, thereby generating a polynucleotide fragment with a 3′ hydroxyl; and   (d) an enzyme capable of extending the polynucleotide fragment with the 3′ hydroxyl in a template independent manner.   
     
     
         76 . The kit according to claim  1 , wherein the enzyme comprising the nonprocessive 3′ to 5′ exonuclease activity does not comprise an endonuclease activity. 
     
     
         77 . The kit according to  claim 75 , wherein the enzyme comprising the nonprocessive 3′ to 5′ exonuclease activity also comprises an endonuclease activity, and wherein the endonuclease activity is minimized or absent. 
     
     
         78 . The kit according to  claim 75 , wherein the enzyme comprising the nonprocessive 3′ to 5′ exonuclease activity is selected from the group consisting of endonuclease 4, exonuclease T, and apurinic/apyrimidinic endonuclease (APE 1). 
     
     
         79 . The kit according to  claim 75 , further comprising a labeled nucleotide, whereby a polynucleotide fragment labeled at the 3′ end is generated. 
     
     
         80 . The kit according to  claim 79 , wherein the labeled nucleotide is selected from the group consisting of a labeled nucleotide triphosphate (NTP), a labeled deoxynucleotide triphosphate (dNTP), and a labeled dideoxynucleotide triphosphate (ddNTP). 
     
     
         81 . The kit according to  claim 79 , wherein the labeled nucleotide is a biotinylated nucleotide. 
     
     
         82 . The kit according to  claim 79 , wherein the labeled nucleotide comprises a fluorophore. 
     
     
         83 . The kit according to  claim 79 , wherein a mixture of labeled and unlabeled nucleotides is used for labeling the polynucleotide fragment. 
     
     
         84 . The kit according to  claim 75 , wherein the non-canonical nucleotide is dUTP and the enzyme capable of cleaving a base portion of at least one non-canonical nucleotide from a polynucleotide is uracil N-glycosylase (UNG). 
     
     
         85 . The kit according to  claim 75 , further comprising instructions for use in a method for producing polynucleotide fragments, the method comprising:
 (a) cleaving a base portion of at least one non-canonical nucleotide from a polynucleotide, wherein the polynucleotide comprises at least one non-canonical nucleotide, wherein the cleaving comprises use of uracil N-glycosylase (UNG), thereby generating an abasic site in the polynucleotide;   (b) fragmenting the phosphodiester backbone of the polynucleotide at the abasic site using N,N′-dimethylethylenediamine (DMED), thereby forming a polynucleotide fragment with a blocked 3′ end;   (c) unblocking the blocked 3′ end of the polynucleotide fragment with an enzyme comprising a nonprocessive 3′ to 5′ exonuclease activity, thereby generating a polynucleotide fragment with a 3′ hydroxyl; and   (d) extending the polynucleotide fragment with the 3′ hydroxyl from the 3′ hydroxyl using terminal deoxynucleotidyl transferase (TdT).   
     
     
         86 . The kit according to  claim 75 , wherein the agent capable of fragmenting the phosphodiester backbone of the polynucleotide at or near the abasic site is N,N′-dimethylethylenediamine (DMED). 
     
     
         87 . The kit according to  claim 75 , wherein the enzyme capable of extending the polynucleotide fragment with the 3′ hydroxyl in a template independent manner is terminal deoxynucleotidyl transferase (TdT). 
     
     
         88 . The kit according to  claim 75 , wherein (a) is UNG; (b) is DMED; (c) is APE 1; and (d) is TdT. 
     
     
         89 . The kit according to  claim 75 , further comprising components for synthesis of a polynucleotide to be fragmented. 
     
     
         90 . The kit according to  claim 90 , wherein the components comprise a primer and/or nucleotides. 
     
     
         91 . The kit according to  claim 91 , wherein the primer comprises a composite primer. 
     
     
         92 . The kit according to  claim 91 , wherein the nucleotides comprise canonical nucleotides. 
     
     
         93 . The kit according to  claim 91 , wherein the nucleotides comprise non-canonical nucleotides. 
     
     
         94 . The kit according to  claim 91 , wherein the nucleotides comprise canonical and non-canonical nucleotides.