METHOD FOR CONTROLLED DNA FRAGMENTATION
First Claim
1. A method for fragmenting nucleic acids from an initial nucleic acid sample in an in vitro reaction, comprising:
- a) providing a plurality of transpososome complexes, which include (i) a plurality of transposases, (ii) 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, (iii) 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;
b) contacting, in a single reaction mixture, the plurality of transpososome complexes with the nucleic acids, under conditions that are suitable for transposing the first and second transposon end sequences into the nucleic acids and fragmenting the nucleic acids, where the nucleic acids includes a first nucleic acid molecule; and
c) producing at least one fragmented tagged 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 transposon end sequences into the first nucleic acid molecule at a first position and fragmenting and tagging the first nucleic acid molecule, and by transposing the second transposon end sequences into first nucleic acid molecule at a second position and fragmenting and tagging the first nucleic acid molecule, wherein the at least one fragmented tagged DNA molecules 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.
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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.
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Citations
20 Claims
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1. A method for fragmenting nucleic acids from an initial nucleic acid sample in an in vitro reaction, comprising:
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a) providing a plurality of transpososome complexes, which include (i) a plurality of transposases, (ii) 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, (iii) 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; b) contacting, in a single reaction mixture, the plurality of transpososome complexes with the nucleic acids, under conditions that are suitable for transposing the first and second transposon end sequences into the nucleic acids and fragmenting the nucleic acids, where the nucleic acids includes a first nucleic acid molecule; and c) producing at least one fragmented tagged 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 transposon end sequences into the first nucleic acid molecule at a first position and fragmenting and tagging the first nucleic acid molecule, and by transposing the second transposon end sequences into first nucleic acid molecule at a second position and fragmenting and tagging the first nucleic acid molecule, wherein the at least one fragmented tagged DNA molecules 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification