Transposon end compositions and methods for modifying nucleic acids
View Patent ↗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).
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.
2. The method of claim 1 , wherein the target nucleic acid comprises genomic DNA.
3. The method of claim 1 , wherein the target nucleic acid comprises cDNA.
4. The method of claim 1 , wherein the transposon end polynucleotides comprise a tag domain selected from the group consisting of a sequencing tag domain, a capture tag domain, and an amplification tag domain.
5. The method of claim 1 , wherein the transposase is selected from a Tn5 transposase, a hyperactive Tn5 transposase, and a Mu transposase.
6. The method of claim 1 , wherein (b) comprises extending the 3′ ends of the nucleic acid fragments.
7. The method of claim 6 , wherein (b) comprises contacting the nucleic acid fragments with a strand-displacing nucleic acid polymerase.
8. The method of claim 6 , wherein (b) comprises contacting the nucleic acid fragments with a nucleic acid polymerase having 5′-to-3′ exonuclease activity.
9. The method of claim 6 , wherein (b) comprises contacting the nucleic acid fragments with a ligase and a nucleic acid polymerase lacking both strand-displacing and 5′-to-3′ exonuclease activities.
10. The method of claim 6 , wherein (b) comprises contacting the nucleic acid fragments with a ligase and a ligation oligonucleotide.
11. The method of claim 1 , wherein the non-selectively amplifying is selected from the group consisting of a strand-displacement amplification reaction, a rolling circle amplification reaction, a loop-mediated amplification reaction, and PCR.
12. The method of claim 1 , wherein (c) comprises capturing the amplified nucleic acid fragments on a surface.
13. The method of claim 12 , wherein the surface comprises at least a million attached nucleic acid fragments.
14. The method of claim 12 , wherein the surface is on a substrate selected from the group consisting of a bead, a chip, a slide, a microtiter plate, a tube, a microchannel, and a dipstick.
15. The method of claim 12 , wherein the surface comprises at least a million captured nucleic acid fragments.
16. The method of claim 1 , further comprising sequencing the library of amplified nucleic acid fragments.
17. A method for preparing a library of nucleic acid fragments 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 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.
18. The method of claim 17 , wherein the library of nucleic acid fragments is representative of nucleic acid sequences of the plurality of nucleic acid fragments comprising one of the transposon end polynucleotides attached to each end of the nucleic acid fragments.
19. The method of claim 17 , wherein the library of nucleic acid fragments lacks nucleic acid sequences of the plurality of nucleic acid fragments comprising one of the transposon end polynucleotides attached to a single end of the nucleic acid fragments.