Method for controlled dna fragmentation

Abstract

A composition and method for controlled in vitro fragmentation of nucleic acids. A transposase forms catalytically active complexes with a modified transposon end that contains within its end sequence degenerate, apurinic/apyrimidinic sites, nicks, or nucleotide gaps, to fragment or shear a target nucleic acid sample in a controlled process. This method yields desired average nucleic acid fragment sizes. The inventive composition and method may be applied for generation of DNA fragments containing shortened transposon end sequences to facilitate subsequent reactions, for production of asymmetrically tailed DNA fragments, etc.

1 - 62 . (canceled) 
     
     
         63 . An in vitro method for fragmenting DNA, comprising:
 a) forming a plurality of transposome complexes by contacting in a single reaction mixture
 (i) a plurality of transposases with 
 (ii) a plurality of polynucleotides containing a first transposon end sequence, wherein the first transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the first transposon end sequence contains at least one nick, gap, apurinic site or apyrimidinic site, and 
 (iii) a plurality of polynucleotides containing a second transposon end sequence, wherein the second transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the second transposon end sequence contains at least one nick, gap, apurinic site or apyrimidinic site; and 
   b) contacting the plurality of transposome complexes with a plurality of target DNA molecules; and   c) producing at least one fragmented DNA molecule having a first end joined to the first transposon end sequence and a second end joined to the second transposon end sequence, by transposing the first and the second transposon end sequences into the target DNA molecules and fragmenting the target DNA, wherein the at least one fragmented DNA molecule includes the first transposon end sequence having at least one nick, gap, apurinic site or apyrimidinic site, and a second end having at least one nick, gap, apurinic site or apyrimidinic site.   
     
     
         64 . The method of  claim 63  wherein the first and second transposon ends are selected from a Mu transposon end, a Mos1 transposon end, a Vibrio harvey transposon end, and a Tn5 transposon end. 
     
     
         65 . The method of  claim 63  wherein the first and second transposase are selected from a MuA transposase, a Mos1 transposase, a Vibrio harvey transposase, and a Tn5 transposase. 
     
     
         66 . The method of  claim 63  further comprising the step of contacting the target DNA fragments comprising the transposon end at the 5′ ends with DNA polymerase having 5′-3′ exonuclease or strand displacement activity, resulting in fully double-stranded DNA from the target DNA fragments. 
     
     
         67 . The method of  claim 63 , further comprising: amplifying the at least one fragmented DNA molecule to produce amplified fragmented DNA molecules. 
     
     
         68 . The method of  claim 67 , further comprising: denaturing the amplified target DNA to produce a plurality of single-stranded fragmented DNA. 
     
     
         69 . The method of  claim 68 , further comprising: immobilizing the plurality of single-stranded fragmented DNA to a support. 
     
     
         70 . The method of  claim 69 , further comprising: sequencing the plurality of single-stranded fragmented DNA which is immobilized to the support with a massively parallel sequencing reaction. 
     
     
         71 . The method of  claim 70 , wherein the massively parallel sequencing reaction comprises providing a surface having an array of a plurality of reaction sites, and the reaction site is operatively linked to a sensor. 
     
     
         72 . The method of  claim 71 , wherein the sensor detects at least one byproduct or cleavage product of a nucleotide incorporation reaction which is selected from a group consisting of hydrogen ions, protons and pyrophosphate groups. 
     
     
         73 . The method of  claim 71 , wherein the sensor comprises an ISFET. 
     
     
         74 . The method of  claim 63 , wherein the first transposon end sequence comprises a double-stranded nucleic acid having a first attacking strand and a first non-attacking strand. 
     
     
         75 . The method of  claim 74 , wherein the first attacking strand includes a first nick or a first gap. 
     
     
         76 . The method of  claim 74 , wherein the first non-attacking strand includes a first nick or a first gap. 
     
     
         77 . The method of  claim 74 , wherein the nick or gap in the first attacking strand is located after the sixth, eighth, tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventh nucleotide from the 3′ end of the first attacking strand. 
     
     
         78 . The method of  claim 63 , wherein the second transposon end sequence comprises a double-stranded nucleic acid having a second attacking strand and a second non-attacking strand. 
     
     
         79 . The method of  claim 78 , wherein the second attacking strand includes a second nick or a second gap. 
     
     
         80 . The method of  claim 78 , wherein the second non-attacking strand includes a second nick or second gap. 
     
     
         81 . The method of  claim 78 , wherein the nick in the second attacking strand is located after the sixth, eighth, tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventh nucleotide from the 3′ end of the second attacking strand. 
     
     
         82 . The method of  claim 63 , wherein the average length of the fragmented DNA molecules in the plurality can be controlled by (i) varying the amount of transpososome complexes which is contacted with the plurality of target DNA, (ii) varying the amount of target DNA which is contacted with the transpososome complexes, (iii) varying the amount of time of the transposition reaction, or (iv) varying the location of the nick or gap on the transposon end sequence.