Single molecule arrays for genetic and chemical analysis

• US 8,445,197 B2
• Filed: 10/31/2007
• 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:
  US11/982,467
• Publication Number:
  US 20090137414A1
• Time in Patent Office: 2,029 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 majority of said plurality of discrete regions comprises a single concatemer comprising a plurality of monomeric units,

wherein each monomeric unit comprises a first target sequence of said target polynucleotide and an adaptor, and said first target sequence is adjacent to said adaptor,

and wherein said substrate is prepared by a photolithography method such that said plurality of discrete regions comprise a functional group for non-covalent attachment of said concatemer;

(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) detected said ligated complex, thereby identifying a nucleotide of said target polynucleotide;

(f) removing said ligated complex;

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

2. The method of claim 1, wherein said substrate is a planar material that comprises silica.

3. The method of claim 1, wherein said functional group comprises an amine group.

4. The method of claim 3, wherein said amine group is an amino group.

5. 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.

6. The method of claim 5, wherein said detectable label comprises a fluorophore.

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

8. 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.

9. 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.

10. The method of claim 9, wherein said generating is through a rolling circle replication reaction.

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

12. The method of claim 1, wherein at least 70% of said plurality of discrete regions comprise a single concatemer.

13. The method of claim 1, wherein said target polynucleotide is human genomic DNA.