Method for preparing single-stranded DNA libraries

• US 20040185484A1
• Filed: 01/28/2004
• Published: 09/23/2004
• Est. Priority Date: 01/29/2003
• Status: Abandoned Application

• Current Assignee: Gina L. Costa, John H. Leamon, Jonathan M. Rothberg, Michael P. Weiner
• Original Assignee: Gina L. Costa, John H. Leamon, Jonathan M. Rothberg, Michael P. Weiner
• Inventors: Weiner, Michael P., Rothberg, Jonathan M., Leamon, John H., Costa, Gina L.
• Application Number:
  US10/767,894
• Publication Number:
  US 20040185484A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/125 : Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2531/125 : Rolling circle
• CPC Class Codes: C12Q 2535/119 : Double strand sequencing
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/149 : Sequential reactions
• CPC Class Codes: C12Q 2547/101 : by confinement to a single tube/container
• CPC Class Codes: C12Q 2547/107 : Use of permeable barriers, e.g. waxes
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• CPC Class Codes: C12Q 2565/119 : based on extraction of label to an organic phase, i.e. partitioning of label between different organic phases
• CPC Class Codes: C12Q 2565/301 : Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: G01N 2021/6484 : Optical fibres
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01R 33/1269 : of molecules labeled with magnetic beads magnetic particles for bio assay G01N33/54326
• CPC Class Codes: Y02P 20/582 : Recycling of unreacted starting or intermediate materials
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to methods of generating single stranded DNA libraries for use in amplification and sequencing reactions. In various aspects, the disclosed methods include: fragmenting DNA; polishing the fragments'"'"' ends; ligating the fragments to universal adaptors; performing strand displacement and extension of the nicked fragments; purifying the double-stranded ligation products; capturing the double-stranded ligation products onto a solid support; and isolating single stranded DNA library fragments, and binding these fragments to another solid support.

Claims

1. A method for clonally isolating a library comprising a plurality of single stranded DNA molecules comprising:

(a) fragmenting large template DNA molecules to generate a plurality of fragmented DNA molecules;

(b) attaching a first or second universal double stranded adaptor to a first end of each fragmented DNA molecule and a first or second universal adaptor to a second end of each fragmented DNA molecule to form a mixture of adaptor ligated DNA molecules;

(c) isolating a plurality of single stranded DNA molecules each comprising a first single stranded universal adaptor and a second single stranded universal adaptor to obtain a library; and

(d) delivering the single stranded DNA molecules into reactors such that a plurality of the reactors include one DNA molecule, thereby clonally isolating the library.

3. The method according to claim 1 or 2, wherein the first universal double stranded adaptor or the second universal adaptor is attached by ligation.

4. The method according to claim 1, wherein (f) is accomplished by:

i) delivering the single stranded DNA molecules onto a location on a reactor array;

or ii) delivering the single stranded DNA molecules into droplets in a water-in-oil emulsion.

5. The method according to claim 1 or 2, further comprising the step of repairing single stranded nicks in the mixture of adaptor ligated DNA molecules using DNA repair and modifying enzymes.

6. The method according to claim 5, wherein the DNA repair and modifying enzymes are selected from the group consisting of polymerase, ligase, kinase, and combinations thereof.

7. The method according to claim 6, wherein the polymerase is Bacillus stearothermophilus polymerase I, the ligase is T4 ligase, and the kinase is T4 polynucleotide kinase.

8. The method according to claim 1 or 2, wherein the template DNA is selected from the group consisting of genomic DNA, cDNA, plasmid DNA, cosmid DNA, artificial chromosome DNA, synthetic DNA, phasemid DNA, and phagemid DNA.

9. The method according to claim 1 or 2, wherein the template DNA comprises reverse transcripts.

10. The method according to claim 1 or 2, wherein the fragmenting is performed by a means selected from the group consisting of enzymatic, chemical, and mechanical means.

11. The method according to claim 10, wherein the enzymatic means is DNase I.

12. The method according to claim 10, wherein the mechanical means is nebulization.

13. The method according to claim 11, wherein the DNase I digestion is performed at a temperature of 10-37°

C. for 1-2 minutes.

14. The method according to claim 10, wherein the enzymatic means is a restriction endonuclease.

15. The method according to claim 10, wherein the mechanical means is selected from the group consisting of a French Press, a sonicator, a HydroShear, and a nebulizer.

16. The method according to claim 1 or 2, wherein the fragmented DNA molecules are 50 bp to 700 bp in length.

17. The method according to claim 1 or 2, wherein the compatible ends are blunt ends.

18. The method according to claim 17 wherein the blunt ends are created with an enzyme selected from the group consisting of Pfu polymerase, T4 DNA polymerase and Klenow fragments.

19. The method according to claim 1 or 2, wherein the compatible ends include an A or T overhang.

20. The method according to claim 1 or 2, wherein the first or second double stranded universal adaptor comprises phosphorothioate linkages.

21. The method according to claim 1 or 2, wherein a biotin moiety is attached to the first or second double stranded universal adaptor.

22. The method according to claim 1 or 2, wherein the first or second double stranded universal adaptor is ligated with T4 DNA ligase.

23. The method according to claim 1 or 2, wherein either the first double stranded universal adaptors or the second double stranded universal adaptors or both double stranded universal adaptors comprise a discriminating key sequence.

24. The method according to claim 23, wherein the discriminating key sequence is 3-12 nucleotides in length.

25. The method according to claim 23, wherein the discriminating key sequence comprises at least one nucleotide selected from the group consisting of A, G, C, U, and T.

26. The method according to claim 1 or 2, wherein first and second double stranded universal adaptor comprise a PCR priming sequence and a sequencing primer sequence.

27. The method according to claim 26, wherein the PCR priming sequence is 10-20 base pairs in length.

28. The method according to claim 26 wherein the sequencing primer sequence is 10-20 base pairs in length.

29. The method according to claim 26, wherein the PCR priming sequence and the sequencing primer sequence overlap.

30. The method according to claim 1 or 2, wherein the mixture of adaptor ligated DNA molecules is separated by a method selected from the group consisting of gel electrophoresis, filtration, size exclusion chromatography, and sucrose sedimentation.

31. The method according to claim 1 or 2, wherein the plurality of single stranded DNA molecules is attached to a DNA capture bead.

32. The method according to claim 31, wherein the DNA capture bead comprises a component of a binding pair.

33. The method according to claim 31 wherein the binding pair is selected from the group consisting of avidin/biotin, ligand/receptor, antigen/antibody and complementary nucleotides.

34. The method according to claim 31, wherein the DNA capture bead is a paramagnetic bead.

35. The method according to claim 1 or 2, wherein the plurality of single stranded DNA molecules is obtained by a treatment selected from the group consisting of low salt treatment, high pH treatment, and chemical denaturation treatment.

55. The method of claim 1, 2 or 49 further comprising the step of attaching the isolated single stranded molecules each individually to a solid support.

2. A method for generating a library comprising a plurality of single stranded DNA molecules, comprising:

(a) fragmenting large or whole genomic template DNA molecules to generate a plurality of fragmented DNA molecules;

(b) ligating a first universal double stranded adaptor or a second universal adaptor to a first end of each fragmented DNA molecule and a first universal adaptor or second universal adaptor to a second end of each fragmented DNA molecule to produce a mixture of adaptor ligated DNA molecules, wherein the first universal adaptor contains a moiety that binds to a solid support;

(c) attaching to a solid support those DNA molecules comprising a first double stranded universal adaptor;

(d) removing adaptor ligated DNA molecules which have not attached to a solid support;

(e) strand separating those adaptor ligated DNA molecules that are attached to a solid support at only one end to release a plurality of single stranded DNA molecules having a first single stranded universal adaptor at one end and a second single stranded adaptor at the other end; and

(f) isolating a library of the released single stranded DNA molecules of step (e) away from those DNA molecules that remain attached to the solid support.

36. A method for generating a single stranded DNA library attached to solid supports comprising:

(a) generating a plurality of single stranded DNA templates;

(b) attaching each of the plurality of ssDNA templates to a solid support; and

(c) isolating the solid supports on which the single stranded DNA templates are attached.

37. A method for generating a single stranded DNA library attached to solid supports comprising:

(a) fragmenting large template DNA molecules to generate a plurality of fragmented DNA molecules;

(b) attaching a first or second universal double stranded adaptor to a first end of each fragmented DNA molecule and a first or second universal adaptor to a second end of each fragmented DNA molecule to make a mixture of adaptor ligated DNA molecules;

(c) isolating those single stranded DNA molecules which comprise a first single stranded universal adaptor and a second single stranded universal adaptor; and

(d) attaching the isolated single stranded molecules from (c) to a solid support.

38. The method according to claim 37, wherein the solid support is a DNA capture bead.

39. The method according to claim 37, wherein the DNA is selected from the group consisting of genomic DNA, cDNA, plasmid DNA, cosmid DNA, artificial chromosome DNA, synthetic DNA, phasemid DNA, and phagemid DNA.

40. The method according to claim 37, wherein the DNA is attached to the solid support via a binding pair.

41. The method according to claim 40, wherein the binding pair is selected from the group consisting of avidin/biotin, ligand/receptor, antigen/antibody and complementary nucleotides.

42. A library of mobile solid supports made by the method of claim 37.

43. A nucleic acid molecule comprising a first adaptor, a fragment of template DNA, and a second adaptor, wherein the first adaptor and second adaptor each comprise a sequencing primer, a PCR primer, and a discriminating key sequence, and wherein the first adaptor and second adaptor, when dissociated, do not cross-hybridize to each other under stringent hybridization conditions.

44. The nucleic acid molecule of claim 43, wherein the PCR primer is 10-20 base pairs in length.

45. The nucleic acid molecule of claim 43, wherein the sequencing primer is 10-20 base pairs in length.

46. The nucleic acid molecule of claim 43, wherein the discriminating key sequence is 3 to 12 base pairs in length.

47. The nucleic acid molecule of claim 43, wherein the template DNA is selected from the group consisting of genomic DNA, cDNA plasmid DNA, cosmid DNA, artificial chromosome DNA, synthetic DNA, phasemid DNA, and phagemid DNA.

48. The nucleic acid molecule of claim 43, wherein the nucleic acid molecule, when dissociated, has minimal cross-hybridization to dissociated template DNA.

49. A method for preparing single stranded DNA molecules, comprising:

(a) fragmenting large or whole genomic template DNA molecules to generate a plurality of fragmented DNA molecules;

(b) ligating a first universal double stranded adaptor or a second universal adaptor to a first end of each fragmented DNA molecule and a first universal adaptor or second universal adaptor to a second end of each fragmented DNA molecule to produce a mixture of adaptor ligated DNA molecules;

(c) attaching adaptor ligated DNA molecules comprising a first double stranded universal adaptor and a second double stranded adaptor to a solid support via one strand of the first double stranded universal adaptor;

(d) washing away adaptor ligated DNA molecules which have not attached to a solid support;

(e) strand separating those adaptor ligated DNA molecules that are attached to a solid support at only one end to release a plurality of single stranded DNA molecules comprising a first single stranded universal adaptor at one end and a second single stranded adaptor at the other end; and

; and

(f) isolating the single stranded DNA molecules.

50. A method for delivering nucleic acid templates to a plurality of reaction centers comprising the steps of:

(a) providing a population of nucleic acid templates;

(b) isolating each nucleic acid template from said population to a sequestering agent to form a population of sequestered nucleic acid templates;

(c) delivering said population of sequestered nucleic acid templates to said plurality of reaction centers wherein each reaction center receives one sequestered nucleic acid.

51. The method of claim 50, wherein said isolating step comprises attaching said nucleic acid templates to a bead.

52. The method of claim 50, wherein said isolating step comprises encapsulating said nucleic acid template in an emulsion of a water-in-oil emulsion.

53. The method of claim 52, wherein said nucleic acid template is encapsulated with a bead and wherein the bead can bind said nucleic acid.

54. The method of claim 50, wherein said delivering step comprises delivering said sequestered nucleic acid to a plurality of reaction centers, wherein each reaction center is a well on a picotiter plate.