Single molecule arrays for genetic and chemical analysis

• US 8,445,194 B2
• Filed: 06/13/2006
• Issued: 05/21/2013
• Est. Priority Date: 06/15/2005
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Drmanac, Radoje, Callow, Matthew J., Drmanac, Snezana, Hauser, Brian K., Yeung, George
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US11/451,691
• Publication Number:
  US 20070099208A1
• Time in Patent Office: 2,534 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• 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 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: Y10S 977/778 within specified host or matrix material, e.g. nanocomposite films
• CPC Class Codes: Y10S 977/789 in array format
• CPC Class Codes: Y10S 977/792 : Nucleic acid array, e.g. human genome array
• CPC Class Codes: Y10S 977/88 : with arrangement, process, or apparatus for testing
• CPC Class Codes: Y10S 977/882 : Assembling of separate components, e.g. by attaching

Abstract

Random arrays of single molecules are provided for carrying out large scale analyses, particularly of biomolecules, such as genomic DNA, cDNAs, proteins, and the like. In one aspect, arrays of the invention comprise concatemers of DNA fragments that are randomly disposed on a regular array of discrete spaced apart regions, such that substantially all such regions contain no more than a single concatemer. Preferably, such regions have areas substantially less than 1 μm² and have nearest neighbor distances that permit optical resolution of on the order of 10⁹ single molecules per cm². Many analytical chemistries can be applied to random arrays of the invention, including sequencing by hybridization chemistries, sequencing by synthesis chemistries, SNP detection chemistries, and the like, to greatly expand the scale and potential applications of such techniques.

Claims

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

a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of said discrete regions, wherein at least a majority of said discrete regions comprises a single concatemer, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, and

wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises;

i) a first target sequence of said target polynucleotide;

ii) an adaptor comprising a Type IIs endonuclease restriction site, wherein said first target sequence is adjacent to said adaptor;

b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor;

c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence;

d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex;

e) detecting said ligated complex thereby identifying a nucleotide of said target polynucleotide;

f) stripping off said ligated complex;

g) repeating steps (b) through (f) in order to determine said sequence of said target polynucleotide.

2. The method of claim 1, wherein one or more probes from said first set or from said second set comprise a detectable label and wherein detecting said ligated complex comprises detecting said detectable label.

3. The method of claim 2, wherein said detectable label comprises a fluorophore.

4. The method of claim 1, wherein said one or more probes from said first set comprise one or more degenerate nucleotides.

5. The method of claim 1, wherein forming said ligated complex produces a detectable signal, and wherein said detecting step (e) comprises detecting said detectable signal.

6. The method of claim 1, wherein one or more probes of said second set of probes comprise one or more degenerate nucleotides.

7. The method of claim 1, wherein said concatemers are formed by a method comprising;

a) providing fragments of said target polynucleotide;

b) ligating an adaptor to a terminus of a plurality of said fragments;

c) circularizing said fragments ligated to said adaptors to form circular products;

d) generating a concatemer from at least one of said circular products, thereby forming said concatemers.

8. The method of claim 7, wherein said generating is accomplished using rolling circle replication.

9. The method of claim 7, wherein said fragments substantially cover said target polynucleotide.

10. The method of claim 1, wherein said Type IIs endonuclease is Mmel or Eco P15l.

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

a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of discrete regions, wherein at least a majority of said discrete regions comprises a single concatemer disposed thereon, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, and

wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises;

i) a first target sequence of said target polynucleotide;

ii) an adaptor, wherein said first target sequence is adjacent to said adaptor;

b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor;

c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence;

d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex;

e) detecting said ligated complex;

thereby identifying said sequence of said target polynucleotide.

12. The method of claim 11, wherein said substrate is formed by a method comprising:

a) providing a support comprising a surface, wherein said surface comprises said plurality of discrete regions;

b) providing a composition comprising a plurality of DNA concatemers, wherein each concatemer comprises a plurality of monomeric units;

c) depositing said composition on said surface, thereby randomly disposing said concatemers on said surface such that a majority of said plurality of discrete regions comprises a concatemer;

d) immobilizing said concatemers on said discrete regions.

13. The method of claim 12, wherein said plurality of discrete regions comprise reactive functionalities and said concatemers comprise functionalities complementary to said reactive functionalities, and wherein said immobilizing step (d) occurs by covalent linkages formed between said reactive functionalities and said complementary functionalities.

14. The method of claim 11 wherein said substrate further comprises inter-regional areas between said discrete regions, and said concatemers do not bind to said inter-regional areas.

15. The method of claim 11 wherein said majority of said concatemers are immobilized on said plurality of discrete regions through a noncovalent interaction.

16. The method of claim 15 wherein said concatemers have a diameter that is approximately equal to that of the discrete regions.

17. The method of claim 12 wherein said majority of said concatemers are immobilized on said plurality of discrete regions through a noncovalent interaction.

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

a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of said discrete regions, wherein a at least majority of said discrete regions comprises a single concatemer, wherein said substrate further comprises inter-regional areas between said discrete regions, wherein said concatemers do not bind to said inter-regional areas, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, and

wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises;

(i) a first target sequence of said target polynucleotide;

(ii) an adaptor, wherein said first target sequence is adjacent to said adaptor;

b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor;

c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence;

d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex;

e) detecting said ligated complex, thereby identifying a nucleotide of said target polynucleotide;

f) stripping off said ligated complex;

g) repeating steps (b) through (f) in order to determine said sequence of said target polynucleotide.

19. The method of claim 18, wherein said majority of said concatemers are immobilized on said plurality of discrete regions through a noncovalent interaction.

20. The method of claim 19 wherein said wherein said inter-regional areas are hydrophobic.