Error-free amplification of DNA for clonal sequencing

• US 20090105094A1
• Filed: 09/26/2008
• Published: 04/23/2009
• Est. Priority Date: 09/28/2007
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Heiner, Cheryl, Travers, Kevin
• Granted Patent: US 8,003,330 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Methods of preparing nucleic acid templates and providing the nucleic acid templates to low copy number reaction volumes are provided. Related compositions of nucleic acid templates are also provided.

Claims

1. A method of providing low copy number circularized nucleic acid variants to reaction volumes, the method comprising:

(a) providing a population of clonal nucleic acids;

(b) generating a set of overlapping nucleic acid fragments from the population of clonal nucleic acids;

(c) circularizing the set of overlapping nucleic acid fragments to produce circularized nucleic acid variants of at least one member of the clonal population, the variants comprising overlapping subsequences of the member; and

,

(d) aliquotting the circularized nucleic acid variants into low copy number reaction volumes.

2. The method of claim 1, wherein (a) comprises:

producing a population of nucleic acid fragments of similar lengths;

attaching tags to ends of the nucleic acid fragments of similar lengths to produce tagged fragments; and

,

amplifying the tagged fragments to generate the population of clonal nucleic acids.

3. The method of claim 2, wherein producing the population of nucleic acid fragments of similar lengths comprises providing a population of nucleic acids whose lengths are within a 20% range of one another.

4. The method of claim 2, wherein producing the population of nucleic acid fragments of similar lengths comprises:

providing a template nucleic acid;

binding the template nucleic acid to a plurality of single-walled carbon nanotubes (CNT) of similar length;

cleaving a portion of the template nucleic acid that is not bound to the CNTs;

separating a CNT-bound portion of the template nucleic acid from the portion of the template nucleic acid that is not bound to the CNTs; and

,

releasing the CNT-bound portion of the template nucleic acid from the CNTs to produce the population of nucleic acid fragments of similar lengths.

5. The method of claim 4, wherein providing the template nucleic acid comprises providing a genomic DNA.

6. The method of claim 5, wherein the genomic DNA has been denatured to produce a single-stranded DNA.

8. The method of claim 4, wherein the template nucleic acid is a cDNA derived from a tissue.

9. The method of claim 4, wherein the template nucleic acid comprises a concatemer that comprises a plurality of tandem short expressed sequence tags (ESTs).

10. The method of claim 2, wherein attaching tags to ends of nucleic acid fragments comprises hybridizing the tags to the ends of the nucleic acid fragments and extending the tags with a polymerase to produce tagged fragments.

11. The method of claim 2, wherein attaching tags to ends of the nucleic acid fragments comprises ligating the tags to the ends of the nucleic acid fragments with a ligase to produce tagged fragments.

12. The method of claim 11, wherein the ligase, which ligates the tags to the ends of the nucleic acid fragments, comprises an enzyme capable of catalyzing intermolecular ligation of single-stranded DNA molecules.

13. The method of claim 2, wherein attaching tags to ends of the nucleic acid fragments comprises attaching tags comprising one or more moieties selected from:

a biotinylated nucleotide, a phosphorylated nucleotide, a methylated nucleotide, a sequence capable of forming a hairpin secondary structure, an oligonucleotide hybridization site, a restriction site, a DNA promoter, an RNA promoter, a sample or library identification tag, and a cis regulatory sequence to the ends of the nucleic acid fragments, thereby forming tagged fragments.

14. The method of claim 2, wherein amplifying the tagged fragments comprises performing:

emulsion PCR, polony amplification, or surface amplification to produce the population of clonal nucleic acids.

15. The method of claim 2, wherein amplifying the tagged fragments comprises amplifying the tagged fragments at an exponential rate to produce the population of clonal nucleic acids.

16. The method of claim 15, wherein amplifying the tagged fragments comprises linear amplification of the tagged fragments prior to amplifying the tagged fragments at an exponential rate, which linear amplification results in a reduction of nucleotide misincorporation as compared to exponential amplification alone, to produce the population of clonal nucleic acids.

17. The method of claim 1, wherein (b) comprises cleaving the population of clonal nucleic acids to produce the set of overlapping nucleic acid fragments.

18. The method of claim 17, wherein cleaving the population of clonal nucleic acids comprises:

enzymatic digestion, sonication, mechanical shearing, electrochemical cleavage, or nebulization to produce the set of overlapping nucleic acid fragments.

19. The method of claim 1, wherein (b) comprises copying overlapping subsequences of the population of clonal nucleic acids to produce the set of overlapping nucleic acid fragments.

20. The method of claim 19, wherein copying overlapping subsequences of the population of clonal nucleic acids comprises extending nested primers, which nested primers hybridize to interior sequences of the clonal nucleic acids, with a polymerase to produce the set of overlapping nucleic acid fragments.

21. The method of claim 1, wherein (c) comprises ligating a first end of a fragment to a second end of the fragment with a ligase to produce circularized variants.

22. The method of claim 21, wherein the ligase which ligates a first end of a fragment to a second end of the fragment to produce circularized variants comprises an enzyme capable of catalyzing intramolecular ligation of single-stranded DNA molecules.

23. The method of claim 1, wherein aliquotting the circularized variants into low copy number reaction volumes comprises flowing or depositing the circularized variants onto a zero-mode waveguide.

24. The method of claim 1, wherein reaction volumes comprise one or more sequencing reagents selected from:

a labeled nucleotide or nucleotide analog, a polymerase, and a divalent cation.

25. The method of claim 1, wherein the steps (a)-(c) are performed in a single reaction vessel.

26. The method of claim 1, wherein the steps (a)-(d) are performed in an in vitro environment, which in vitro environment is an environment outside of a living organism.

31. The method of claim 26, wherein an in vitro environment comprises an environment outside of a living organism.

7. (canceled)

27. A method of producing and flowing or depositing circularized single-stranded DNA variants onto a zero-mode waveguide, which method is performed in a single reaction vessel, the method comprising:

providing a population of single-stranded DNA fragments of similar lengths;

attaching tags to ends of the single-stranded DNA fragments of similar size, wherein attaching the tags to the ends of the single-stranded DNA fragments comprises ligating the tags to the ends of the fragments with an enzyme capable of catalyzing intermolecular ligation of single-stranded DNA molecules to form tagged fragments;

amplifying the tagged fragments at a linear rate to produce a pool of amplified tagged fragments;

amplifying the pool of amplified tagged fragments at an exponential rate to produce a set of clonal tagged single-stranded DNAs;

generating overlapping tagged single-stranded DNA fragments from the set of clonal tagged single-stranded DNAs;

circularizing the overlapping tagged single-stranded DNA fragments, wherein circularizing the fragments comprises ligating a first end of a fragment to a second end of the fragment with an enzyme capable of catalyzing intramolecular ligation of single-stranded DNA molecules to produce circularized single-stranded DNA variants of at least one member of the clonal population, the variants comprising overlapping sequences of the member; and

,

flowing or depositing the circularized single-stranded DNA variants onto a zero-mode waveguide.

28. A method of providing a set of overlapping nucleic acid fragments, the method comprising:

(a) providing a template nucleic acid;

(b) fragmenting the template nucleic acid to form nucleic acid fragments in vitro;

(c) amplifying the nucleic acid fragments to produce a population of clonal nucleic acids in vitro; and

,

(d) generating a set of overlapping nucleic acid fragments from the population of clonal nucleic acids to produce a set of overlapping nucleic acid fragments in vitro.

29. The method of claim 28, wherein the method comprises circularizing the overlapping nucleic acid fragments in vitro to produce circularized variants of at least one member of the clonal population, the variants comprising overlapping sequences of the member.

30. The method of claim 28, wherein steps (a)-(d) are performed in a single reaction vessel.

32. A composition comprising a plurality of single-stranded circularized nucleic acid segments of a nucleic acid, wherein the segments of the set comprise overlapping subsequences of the nucleic acid.

33. The composition of claim 32, wherein the composition comprises a ligase enzyme.

34. The composition of claim 33, wherein the ligase comprises an enzyme capable of catalyzing intramolecular ligation of single-stranded DNA molecules.

35. The composition of claim 32, wherein the nucleic acid comprises a DNA.