Transposon end compositions and methods for modifying nucleic acids
DCFirst Claim
1. A method for preparing a library of nucleic acid fragments representative of a target nucleic acid comprising:
- (a) contacting a target nucleic acid with a plurality of transposomes, wherein the target nucleic acid is double-stranded DNA, and the transposomes comprise a transposase and a pair of transposon end polynucleotides;
(b) incubating the target nucleic acid and transposomes under conditions whereby the target nucleic acid is fragmented into a plurality of nucleic acid fragments comprising one of the transposon end polynucleotides attached to an end of the nucleic acid fragments; and
(c) non-selectively amplifying the nucleic acid fragments, thereby obtaining a library of nucleic acid fragments that is representative of the target nucleic acid from which the fragments were generated.
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Abstract
The present invention provides methods, compositions and kits for using a transposase and a transposon end for generating extensive fragmentation and 5′-tagging of double-stranded target DNA in vitro, then using a DNA polymerase for generating 5′- and 3′-tagged single-stranded DNA fragments without performing a PCR amplification reaction, wherein the first tag on the 5′-ends exhibits the sequence of the transferred transposon end and optionally, an additional arbitrary sequence, and the second tag on the 3′-ends exhibits a different sequence from the sequence exhibited by the first tag. The method is useful for generating 5′- and 3′-tagged DNA fragments for use in a variety of processes, including processes for metagenomic analysis of DNA in environmental samples, copy number variation (CNV) analysis of DNA, and comparative genomic sequencing (CGS), including massively parallel DNA sequencing (so-called “next generation sequencing).
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Citations
19 Claims
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1. A method for preparing a library of nucleic acid fragments representative of a target nucleic acid comprising:
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(a) contacting a target nucleic acid with a plurality of transposomes, wherein the target nucleic acid is double-stranded DNA, and the transposomes comprise a transposase and a pair of transposon end polynucleotides; (b) incubating the target nucleic acid and transposomes under conditions whereby the target nucleic acid is fragmented into a plurality of nucleic acid fragments comprising one of the transposon end polynucleotides attached to an end of the nucleic acid fragments; and (c) non-selectively amplifying the nucleic acid fragments, thereby obtaining a library of nucleic acid fragments that is representative of the target nucleic acid from which the fragments were generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for preparing a library of nucleic acid fragments comprising:
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(a) contacting a target nucleic acid with a plurality of transposomes, wherein the target nucleic acid is double-stranded DNA, and the transposomes comprise a transposase and a pair of transposon end polynucleotides; (b) incubating the target nucleic acid and transposomes under conditions whereby the target nucleic acid is fragmented into a plurality of nucleic acid fragments comprising one of the transposon end polynucleotides attached to at least one end of the nucleic acid fragments; and (c) non-selectively amplifying the nucleic acid fragments, thereby obtaining a library of nucleic acid fragments. - View Dependent Claims (18, 19)
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Specification