Single molecule arrays for genetic and chemical analysis

• US 8,445,196 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 T., Callow, Matthew J., Drmanac, Snezana, Hauser, Brian K., Yeung, George
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US11/981,767
• Publication Number:
  US 20090137404A1
• 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 random array of concatemers, wherein said random array comprises:

(a) a solid substrate comprising a surface, wherein said surface comprises a plurality of discrete spaced apart regions defined on said surface, wherein said discrete spaced apart regions are separated by inter-regional areas;

(b) concatemers randomly disposed on said plurality of discrete spaced apart regions such that at least a majority of said plurality of discrete spaced apart regions comprises a single concatemer immobilized thereon and said concatemers do not bind to said inter-regional areas,

wherein said concatemers comprise a plurality of monomeric units and each monomeric unit comprises;

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

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

wherein each said single concatemer is immobilized on a discrete spaced apart region by attractive noncovalent interactions between said discrete spaced apart region and multiple monomeric units of said single concatemer.

2. The random array of claim 1, wherein said plurality of concatemers are immobilized on said discrete spaced apart regions through attractive noncovalent interactions.

3. The random array of claim 2, wherein said attractive noncovalent interactions are a member selected from Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions.

4. The random array of claim 1, wherein said surface comprises silica.

5. The random array of claim 4, wherein said discrete spaced apart regions comprise functional moieties.

6. The random array of claim 5, wherein said functional moieties comprise amines.

7. The random array of claim 1, wherein said adaptor further comprises a palindrome.

8. The random array of claim 1, wherein a majority of said concatemers disposed on said discrete spaced apart regions have a nearest neighbor distance of about 50 nanometers or greater.

9. The random array of claim 8, wherein said majority of said concatemers have a nearest neighbor distance of about 200 nanometers or greater.

10. The random array of claim 1, wherein said majority of said concatemers are disposed on said discrete spaced apart regions at a density of at least 1000 per μ

m².

19. The random array of claim 1, 11, or 15, wherein said discrete spaced apart regions are prepared by photolithography.

20. The random array of claim 1, 11, or 15, wherein said inert interregional areas comprise hydrophobic barriers.

11. A random array of concatemers, wherein said random array comprises:

(a) a solid substrate comprising a surface, wherein said surface comprises a plurality of discrete spaced apart regions defined on said surface, wherein said discrete spaced apart regions are separated by inter-regional areas;

(b) concatemers randomly disposed on said plurality of discrete spaced apart regions such that at least a majority of said discrete spaced apart regions comprises a single concatemer immobilized thereon and said concatemers do not bind to said inter-regional areas, wherein said concatemers are immobilized on said discrete spaced apart regions through attractive noncovalent interactions, and

wherein said concatemers comprise a plurality of monomeric units and each monomeric unit comprises;

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

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

wherein each said single concatemer is immobilized on a discrete spaced apart region by attractive noncovalent interactions between said discrete spaced apart region and multiple monomeric units of said single concatemer.

12. The random array of claim 11, wherein said attractive noncovalent interactions are a member selected from Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions.

13. The random array of claim 11, wherein said discrete spaced apart regions comprise functional moieties.

14. The random array of claim 13, wherein said functional moieties comprise amines.

21. The random array of claim 11, wherein said discrete spaced apart regions are arranged on said surface in a rectilinear pattern.

15. A random array of concatemers, wherein said random array comprises:

(a) a solid substrate comprising a surface, wherein said surface comprises a plurality of discrete spaced apart regions defined on said surface, wherein said discrete spaced apart regions are separated by inter-regional area;

(b) concatemers randomly disposed on said plurality of discrete spaced apart regions such that at least a-majority of said plurality of discrete spaced apart regions comprises a single concatemer immobilized thereon and said concatemers do not bind to said interregional areas, wherein said concatemers are immobilized on said discrete spaced apart regions through attractive noncovalent interactions, and

wherein said concatemers comprise a plurality of monomeric units and each monomeric unit comprises;

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

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

wherein each said single concatemer is immobilized on a discrete spaced apart region by attractive noncovalent interactions between said discrete spaced apart region and multiple monomeric units of said single concatemer.

16. The random array of claim 15, wherein said concatemers are generated from a circular template, wherein said circular template comprises said adaptor and said first target sequence.

17. The random array of claim 15, wherein said target polynucleotide is a genomic sequence.

18. The random array of claim 15, wherein substantially all of said plurality of discrete spaced apart regions have at most a single concatemer attached.