EFFICIENT ARRAYS OF AMPLIFIED POLYNUCLEOTIDES

• US 20080318796A1
• Filed: 10/29/2007
• Published: 12/25/2008
• Est. Priority Date: 10/27/2006
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Callow, Matthew, DRMANAC, Radoje T.
• Granted Patent: US 7,910,354 B2
• Time in Patent Office: Days
• US Class Current: 506/3
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are arrays of polynucleotides in which the polynucleotides have undergone multiple rounds of amplification in order to increase the strength of signals associated with single polynucleotide molecules.

Claims

1. A method of making a random array of amplified polynucleotides, said method comprising the steps of:

a) providing;

i) a surface, which comprises capture probes, wherein said capture probes have free 3′

ends, and

ii) a plurality of target polynucleotide concatemers disposed on said surface, wherein each concatemer is bound to capture probes at a specific position on said surface; and

b) extending said capture probes such that said concatemers disposed on said surface are amplified;

wherein the amplification products of said concatemers are attached to the surface at or near the specific position of the concatemer amplified, thereby making said random array of amplified polynucleotides.

2. The method of claim 1, wherein amplification products of each said concatemers are attached to said surface through duplexes formed between said concatemers and said capture probes.

7. The method of claim 1, wherein the capture probes are extended using a strand displacement polymerase.

12. An array made according to the method of claim 1.

13. A kit containing the array of claim 12.

3. The method of claim [0006], wherein said concatemers comprise multiple copies of individual target polynucleotides.

4. The method of claim 3, wherein each concatemer comprises multiple copies of a single individual polynucleotide and an adaptor.

5. The method of claim 3, wherein concatemers comprise multiple copies of different individual target polynucleotides, each individual target polynucleotide associated with an adaptor.

6. The method of claim 4, wherein said adaptor comprises a recognition site for a nicking endonuclease, and wherein said recognition site is oriented such that a cleavage site for said nicking endonuclease is located substantially near one end of said adaptor.

17. The method of claim [0006]4, wherein said concatemers comprise multiple copies of individual target polynucleotides.

8. The method of claim [0006], wherein said surface is a planar surface that comprises discrete regions, wherein each discrete region comprises said capture probes attached thereto, and wherein at least a majority of said discrete spaced regions have amplification products of a single concatemer attached.

9. The method of claim 8, wherein each of said discrete regions has an area less than 1 μ

m².

10. The method of claim 8, wherein said discrete regions have a nearest neighbor distance such that a majority of said discrete regions is optically resolvable.

11. The method of claim 8, wherein each of said discrete regions is surrounded by an inter-regional space that is inert.

14. A method of making a random array of target polynucleotides, said method comprising the steps of:

a) providing a plurality of concatemers on a surface, wherein;

i) said surface comprises capture probes,

ii) each of said plurality of concatemers comprises multiple copies of a target polynucleotide and an adaptor; and

iii) each concatemer is attached to a specific position on said surface through duplexes formed between said capture probes and said adaptors in the concatemer, wherein said duplexes comprise a recognition site for a nicking endonuclease;

b) cleaving at least a portion of the plurality of concatemers with a nicking endonuclease to form cleavage products, wherein said cleavage products remain attached to said capture probes;

c) circularizing said cleavage products on said capture probes; and

d) extending said capture probes by rolling circle replication to create at least one copy of each circularized cleavage product;

thereby making said random array of target polynucleotides.

15. An array made according to the method of claim 14.

16. A kit containing the array of claim 15.

18. The method of claim 14, wherein each concatemer comprises multiple copies of a single individual polynucleotide and an adaptor.

19. A method of making a random array of amplified target polynucleotides, said method comprising the steps of:

a) providing a plurality of tailed concatemers;

b) extending said tailed concatemers with a strand-displacing polymerase to form concatemer-extension product complexes; and

c) disposing said concatemer-extension product complexes on a surface, wherein;

i) said surface comprises capture probes,

ii) a majority of said concatemer-extension product complexes from a single concatemer occupy a single region of said surface, and

iii) said majority of said concatemer-extension product complexes is attached to said surface by one or more duplexes formed between said capture probes and said tail portions,

thereby forming said random array of target polynucleotides.

20. The method of claim 19, wherein said tailed concatemers are formed by a method comprising combining a plurality of tailed primers with a plurality of concatemers under annealing conditions, wherein:

a) each of said plurality of concatemers comprises multiple copies of a target sequence and an adaptor;

b) at least a portion of said tailed primers anneal to at least a portion of one or more adaptors; and

c) each of said plurality of tailed primers comprise a tail portion which is not complementary to said concatemers.

21. The method of claim 19, further comprising subsequent to step (c) a step of:

d) extending said majority of said concatemer-extension product complexes with a strand-displacing polymerase.

22. The method of claim 19, wherein the extension products are additionally attached to the surface using chemical attachment.

23. The method of claim 19, wherein the extension products are additionally attached to the surface using hybridization to other capture probe.

24. An array made according to the method of claim 19.

25. A kit containing the array of claim 24.

26. A method of making a random array of target polynucleotides, said method comprising the steps of:

a) combining under annealing conditions a plurality of dendrimers and a plurality of single stranded DNA circles, wherein;

i) each of said plurality of single stranded DNA circles comprises a target polynucleotide and an adaptor, and

ii) each of said plurality of dendrimers comprises a primer capable of annealing to said adaptors of said plurality of single stranded DNA circles,

b) annealing said adaptors of said single stranded DNA circles to said primers of said plurality of dendrimers;

c) extending said primers annealed with said adaptors with a strand-displacing polymerase to form a plurality of dendrimer-extension product complexes; and

d) disposing said dendrimer-extension product complexes on a surface such that each of at least a majority of said plurality of dendrimer-extension product complexes occupies a separate region of said surface,

thereby forming said random array of target polynucleotides.

27. The method of claim 26, wherein said adaptors of different single stranded DNA circles are different.

28. The method of claim 26, wherein each dendrimer-extension product complex comprises multiple different target DNAs.

29. The method of claim 26, wherein:

a) said surface comprises capture probes;

b) each of said plurality of dendrimers further comprises at least one probe complementary to said capture probes; and

c) said disposing comprises attaching said dendrimer-extension product complexes to said surface by forming duplexes between said capture probes and said complementary probes of said dendrimers.

30. An array made according to the method of claim 26.

31. A kit containing the array of claim 30.

32. A method of forming a spatially-compact single stranded amplicon, said method comprising the steps of:

a) combining under annealing conditions a dendrimer and a single stranded DNA circle, wherein;

i) said single stranded DNA circle comprises a target polynucleotide and an adaptor, and

ii) said dendrimer comprises a primer capable of annealing to said adaptor and at least one capture sequence identical to a portion of said single stranded DNA circle,

b) extending said primer annealed to said adaptor with a strand-displacing polymerase to form a single-stranded amplicon, wherein said at least one capture sequence forms a duplex with a complementary portion of said single stranded amplicon,

thereby forming said spatially-compact single stranded amplicon.

33. The method of claim 32, wherein said at least one primer has a free 3′

end.

34. The method of claim 32, wherein the single-stranded amplicon comprises a concatemer of the concatemer of said target polynucleotide and adapter is generated in step b).

35. The method of claim 32, further comprising the step:

c) extending said at least one capture sequence.

36. A plurality of amplicons made from different target polynucleotides according to claim 32.

37. A method of forming a single-stranded amplicon comprising multiple target polynucleotide sequences, said method comprising the steps of:

a) combining under annealing conditions a dendrimer and a plurality of single stranded DNA circles, wherein;

i) each single stranded DNA circle comprises a target polynucleotide and an adaptor, and

ii) said dendrimer comprises multiple sites complementary to adaptors on different DNA circles,

b) extending said primer annealed to said adaptor with a strand-displacing polymerase to form a single stranded amplicon, wherein portions of each amplicon are complementary to target polynucleotides of different DNA circles,

thereby forming said spatially compact single stranded amplicon comprising multiple target polynucleotide sequences.

38. A plurality of amplicons made from different target polynucleotides according to claim 37.

39. A method of forming a double stranded amplicon, the method comprising the steps of:

a) providing a single stranded amplicon comprising a concatemer having multiple copies of a target polynucleotide and an adaptor;

b) annealing primers to the adaptors of the single stranded amplicon; and

c) extending the primers with a non-strand displacing polymerase so that substantially every annealed primer is extended to form an extension product that abuts the next annealed primer, thereby forming a double stranded amplicon.

40. The method of claim 39, further including the step of ligating said extension products to said abutting annealed primers.

41. A method of making a random array of target polynucleotides, said method comprising the steps of:

a) providing a support having a surface;

b) combining in a reaction mixture first primer probes, beads comprising second primer probes on their surfaces, and a plurality of concatemers each comprising multiple copies of a target polynucleotide and an adaptor, said first primer and second primer probes being capable of amplifying in a polymerase chain reaction a portion of said concatemers;

c) forming an emulsion with said reaction mixture so that aqueous compartments are formed that contain second primers, and no more than one bead and no more than one concatemer;

d) conducting a polymerase chain reaction in said aqueous compartments so that portions of said concatemer are amplified on said beads; and

e) disposing said beads from said emulsion onto said surface such that substantially every bead occupies a separate region of said surface,

thereby forming said random array of target polynucleotides.

42. The method of claim 41, wherein said surface of said support has capture probes attached thereto and wherein said bead further comprises at least one probe complementary to the capture probes and wherein said step of disposing further includes attaching said beads to said surface by one or more duplexes formed between capture probes and complements thereof on said beads.

43. A method of identifying a nucleotide sequence of a target polynucleotide, said method comprising:

a) providing a random array comprising a plurality of concatemers disposed on a surface, wherein said concatemers comprise at least one fragment of said target polynucleotide, and wherein said concatemers have undergone at least one round of in situ amplification;

b) hybridizing one or more probes from a first set of probes to said random array under conditions that permit formation of perfectly matched duplexes between said one or more probes from said first set of probes and complementary sequences on said concatemers;

c) hybridizing one or more probes from a second set of probes to each random array under conditions that permit the formation of perfectly matched duplexes between said one or more probes from said second set of probes and complementary sequences on said concatemers;

d) ligating probes from said first and second sets of probes which are hybridized to contiguous sites of said target concatemers;

e) identifying sequences of said ligated probes, thereby generating a sequence read;

f) repeating steps (b) through (e) a number of times to generate multiple sequence reads;

g) assembling said multiple sequence reads,

thereby identifying said nucleotide sequence of said target polynucleotide.

44. A method of identifying a nucleotide sequence of a target polynucleotide, said method comprising:

a) providing a random array comprising a plurality of concatemers disposed on a surface, wherein;

i) said concatemers comprise at least one fragment of said target polynucleotide,

ii) said concatemers further comprise at least one interspersed adaptors, wherein said at least one interspersed adaptor is adjacent to said at least one fragment, and

iii) said concatemers have undergone at least one round of in situ amplification;

b) identifying a sequence of at least a portion of said at least one fragment adjacent to said at least one interspersed adaptor;

thereby identifying said nucleotide sequence of said target polynucleotide.

45. A method of identifying a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:

a) providing a plurality of concatemers, wherein each concatemer comprises a plurality of monomers and each monomer comprises;

i) a first target domain of said target sequence comprising a first set of target detection positions;

ii) a first adaptor comprising a Type IIs endonuclease restriction site;

iii) a second target domain of said target sequence comprising a second set of target detection positions; and

iv) an interspersed adaptor comprising a Type IIs endonuclease restriction site;

b) amplifying said plurality of concatemers;

c) identifying said first nucleotide.

46. The method of claim 45, wherein said plurality of concatemers is immobilized on a surface, and wherein said amplifying comprises in situ amplification of said plurality of concatemers immobilized on said surface.

47. The method of claim 45, wherein said identifying comprises:

a) contacting said concatemers with a set of sequencing probes, wherein each of said set of sequencing probes comprises;

i) a first domain complementary to one of said adaptors;

ii) a unique nucleotide at a first interrogation position; and

iii) a label;

 

wherein if said unique nucleotide is complementary to said first nucleotide, a sequencing probe hybridizes to said concatemer; and

b) identifying said first nucleotide.

48. The method of claim 45, wherein each adaptor comprises an anchor probe hybridization site, and said identifying comprises:

a) hybridizing anchor probes to said anchor probe hybridization sites;

b) hybridizing sequencing probes to target detection positions adjacent to said adaptors;

c) ligating adjacent hybridized sequencing and anchor probes to form ligated probes; and

d) detecting said ligated probes to identify said first nucleotide.

49. The method of claim 45, wherein each adaptor comprises an anchor probe hybridization site, and said identifying comprises:

a) hybridizing anchor probes to said anchor probe hybridization sites;

b) adding a polymerase and at least one dNTP comprising a label, under conditions whereby if said dNTP is perfectly complementary to a detection position, said dNTP is added to the anchor probe to form an extended probe, thereby creating an interrogation position of said extended probe; and

c) identifying a nucleotide at said interrogation position of said extended probe, thereby identifying said first nucleotide at said detection position.

50. The method of claim 45, wherein said surface is functionalized.

51. The method of claim 45, wherein said functionalized surface comprises functional moieties selected from the group consisting of amines, silanes, and hydroxyls.

52. The method of claim 45, wherein said surface comprises a plurality of spatially distinct regions.

53. The method of claim 45, wherein said concatemers are immobilized on said surface using capture probes.

54. The method of claim 45, further comprising fragmenting genomic nucleic acid to form said target sequences.

55. The method of claim 45, wherein said Type IIs endonuclease restriction sites of said first and second adaptors are the same.

56. The method of claim 45, wherein said Type IIs endonuclease restriction sites of said first and second adaptors are different.

57. A random array of polynucleotides, wherein said random array comprises:

a) a substrate having a surface; and

b) a plurality of concatemer extension products disposed on said surface, wherein;

i) said plurality of concatemer extension products is formed from in situ amplification of immobilized concatemers, and

ii) each of said plurality of concatemer extension products comprises at least two copies of a single-stranded concatemer comprising multiple copies of an identical polynucleotide sequence.

58. The random array of claim 57, wherein:

a) said surface comprises capture probes, and

b) said plurality of concatemer extension products are immobilized to said surface through duplexes formed between said concatemer extension products and said capture probes.

59. The random array of claim 57, wherein said capture probes are distributed in discrete regions on said surface, and wherein areas surrounding said discrete regions are inert.

60. The random array of claim 57, wherein at least a portion of said plurality of concatemer extension products comprise target sequences, wherein said target sequences have been identified in a sequencing reaction.

61. A random array of target polynucleotides of unknown sequence, wherein said random array comprises:

a) a substrate having a surface; and

b) a plurality of concatemer extension products disposed on said surface, wherein;

i) said plurality of concatemer extension products is formed from in situ amplification of immobilized concatemers, and

ii) each of said plurality of concatemer extension products comprises at least two copies of a single-stranded concatemer comprising multiple copies of an identical target polynucleotide of unknown sequence.

62. The random array of claim 61, wherein:

a) said surface comprises capture probes, and

b) said plurality of concatemer extension products are immobilized to said surface through duplexes formed between said concatemer extension products and said capture probes.

63. The random array of claim 61, wherein said capture probes are distributed in discrete regions on said surface, and wherein areas surrounding said discrete regions are inert.

64. The random array of claim 61, wherein at least a portion of said plurality of concatemer extension products comprise target sequences, wherein said target sequences have been identified in a sequencing reaction.

65. A kit for in situ amplification, wherein said kit comprises:

a) a support having a surface which comprises first stage amplicons;

b) adaptors and at least one ligase;

c) at least one nicking endonuclease and reactants for a reaction using said at least one nicking endonuclease; and

d) at least one polymerase and reactants for a synthesis reaction using said at least one is said polymerase.