TRANSPOSON END COMPOSITIONS AND METHODS FOR MODIFYING NUCLEIC ACIDS

• US 20100120098A1
• Filed: 10/24/2009
• Published: 05/13/2010
• Est. Priority Date: 10/24/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Epicentre Technologies Corporation (Illumina Incorporated)
• Inventors: Grunenwald, Haiying Li, Caruccio, Nicholas, Dahl, Gary, Jendrisak, Jerome
• Granted Patent: US 9,080,211 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

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.)

Claims

1. A method of tagging a fragment of a target DNA, comprising:

i) incubating target DNA with a transposase and a transposon end or transposon end composition comprising a transferred strand comprising a tag domain and a 3′

portion comprising a transferred strand sequence, under conditions wherein a transposition reaction is catalyzed by said transposase, wherein;

a) said target DNA is fragmented; and

b) said transferred strand of said transposon end or transposon end composition is joined to a 5′

end of a fragment of said target DNA to produce a 5′

-tagged target DNA fragment; and

ii) incubating said 5′

tagged target DNA fragment with a nucleic acid modifying enzyme under conditions wherein a 3′

tag is joined to a 3′

end of said 5′

tagged target DNA fragment to produce a di-tagged target DNA fragment.

2. The method of claim 1, wherein said tagging a fragment comprises generating a library of tagged DNA fragments of a target DNA, wherein said target DNA is fragmented to generate a plurality of target DNA fragments and a transferred strand of said transposon end or transposon end composition is joined to the 5′

ends of each of a plurality of said target DNA fragments to produce a plurality of 5′

tagged target DNA fragments, and wherein said plurality of 5′

-tagged target DNA fragments are incubated with said at least one nucleic acid modifying enzyme under conditions wherein a 3′

tag is joined to a 3′

end of said plurality of 5′

-tagged target DNA fragments to produce a DNA fragment library comprising di-tagged target DNA fragments.

3. The method of claim 1, wherein said nucleic acid modifying enzyme is selected from a group consisting of a template-dependent DNA polymerase, a template-independent DNA polymerase, a template-dependent ligase and a template-independent ligase.

4. The method of claim 3, wherein said nucleic acid modifying enzyme is a template-dependent DNA polymerase that has strand-displacement and/or 5′

nuclease activity.

5. The method of claim 1, wherein said tag domain comprises one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, an address tag domain, and a transcription promoter domain.

7. The method of claim 5, wherein said tag domains are sequencing tag domains that comprise or consist of sequencing tags selected from Roche 454A and 454B sequencing tags, ILLUMINA™

SOLEXA™

sequencing tags, Applied Biosystems'"'"' SOLID™

sequencing tags, the Pacific Biosciences'"'"' SMRT™

sequencing tags, Pollonator Polony sequencing tags, or the Complete Genomics sequencing tags.

8. The method of claim 1, wherein said transposon end composition comprises a hairpin transposon end composition, said hairpin transposon end composition comprising an oligonucleotide that exhibits a non-transferred strand sequence at its 5′

end, a transferred strand sequence at its 3′

end, and an intervening loop sequence comprising a tag domain.

9. The method of claim 1, further comprising amplifying one or more di-tagged target DNA fragments, wherein said amplifying comprises use of one or more of a PCR amplification reaction, a strand-displacement amplification reaction, a rolling circle amplification reaction, a ligase chain reaction, a transcription-mediated amplification reaction, or a loop-mediated amplification reaction.

10. The method of claim 9, wherein said amplifying one or more di-tagged target DNA fragments comprises amplifying a DNA fragment library, wherein said amplifying comprises non-selectively amplifying.

11. The method of claim 9, wherein said amplifying comprises a polymerase chain reaction using a first and a second oligonucleotide primer, each comprising 3′

end portions, wherein at least the 3′

end portion of said first PCR primer is complementary to said 3′

tag of said di-tagged target DNA fragments, and wherein at least a 3′

-end portion of said second PCR primer exhibits the sequence of at least a portion of the 5′

tag or tag domain of said di-tagged target DNA fragments.

12. The method of claim 11, wherein said 5′

end portions of the first and/or said second PCR primers exhibit tag domains, wherein said tag domains comprise one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, an address tag domain, and a transcription promoter domain.

13. The method of claim 12, wherein said tag domains are sequencing tag domains that comprise or consist of sequencing tags selected from Roche 454A and 454B sequencing tags, ILLUMINA™

SOLEXA™

sequencing tags, Applied Biosystems'"'"' SOLID™

sequencing tags, the Pacific Biosciences'"'"' SMRT™

sequencing tags, Pollonator Polony sequencing tags, or the Complete Genomics sequencing tags.

6. (canceled)

14. A composition comprising a synthetic nucleic acid molecule having a 5′

portion comprising a tag domain and a 3′

portion comprising a transferred strand of a transposon end, wherein said tag domain comprises one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, an address tag domain, and a transcription promoter domain.

15. The composition of claim 14, wherein said tag domains comprise a sequencing tags comprising or consisting of sequencing tags selected from Roche 454A and 454B sequencing tags, ILLUMINA™

SOLEXA™

sequencing tags, Applied Biosystems'"'"' SOLID™

sequencing tags, the Pacific Biosciences'"'"' SMRT™

sequencing tags, Pollonator Polony sequencing tags, or the Complete Genomics sequencing tags.

16. The composition of claim 14, further comprising a purified transposase.

17. The composition of claim 16, further comprising a non-ionic detergent.

18. A kit comprising the composition of claim 16.

19. The kit of claim 18, further comprising one or more of a DNA ligase and a DNA polymerase.

20. The kit of claim 18, further comprising reagents for an amplification reaction, wherein said reagents for an amplification reaction comprise at least one primer.

21. The kit of claim 20, wherein said at least one primer comprises at least one sequencing tag selected from the selected from a Roche 454A and 454B sequencing tag, an ILLUMINA™

SOLEXA™

sequencing tag, an Applied Biosystems'"'"' SOLID™

sequencing tag, a Pacific Biosciences'"'"' SMRT™

sequencing tag, a Pollonator Polony sequencing tag, or a Complete Genomics sequencing tag.