Methods for making single molecule arrays

• US 8,133,719 B2
• Filed: 10/31/2007
• Issued: 03/13/2012
• 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
• Application Number:
  US11/981,607
• Publication Number:
  US 20080234136A1
• Time in Patent Office: 1,595 Days
• Field of Search: None
• US Class Current: 435/287.2
• 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 for making an array of randomly disposed concatemers, wherein said method comprises:

(a) providing a solid substrate comprising a surface and a plurality of discrete spaced apart regions defined on said surface,

wherein the discrete spaced apart regions are arranged on the surface in a regular array,

wherein the discrete spaced apart regions bind DNA concatemers through attractive noncovalent interactions,

wherein said discrete spaced apart regions are separated by inter-regional areas; and

said inter-regional areas are hydrophobic;

(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 concatemers on said plurality of discrete spaced apart regions such that at least a majority of said plurality of discrete spaced apart regions comprise a single concatemer bound thereto and said concatemers do not bind to said inter-regional areas.

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

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

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

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

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

7. The method of claim 1, wherein the discrete spaced apart regions are positively charged so as to bind said concatemers.

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

10. The method 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.

11. The method of claim 1 wherein more than 80% of said discrete regions have a single concatemer attached thereto.

12. The method of claim 1, wherein said concatemers have a diameter that is approximately equal to that of the discrete regions.

13. The method of claim 1 wherein said concatemers are made by rolling circle replication.

14. The method of claim 13 wherein the concatemer consists essentially of a polymer of deoxyribonucleotides.

15. The method of claim 1, further comprising the step of determining at least a portion of the DNA sequence of at least one said attached concatemer.

26. The method of claim 1, 16, or 20, wherein said inert inter-regional areas comprise hydrophobic barriers.

27. The method of claim 1, 16, or 20, wherein said discrete spaced apart regions are prepared by photolithography.

28. The method of claim 1, 16, or 20, wherein said target polynucleotide is of unknown sequence.

16. A method for making an array of randomly disposed concatemers, wherein said method comprises:

(a) providing 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 arranged on said surface in a regular array and separated by inter-regional areas;

(b) depositing, on said surface, a composition comprising a plurality of DNA concatemers, wherein said concatemers bind at said discrete spaced apart regions,

thereby randomly disposing concatemers on said plurality of discrete spaced apart regions,

wherein said concatemers are immobilized on said discrete spaced apart regions through attractive noncovalent interactions such that at least a majority of said plurality of discrete spaced apart regions comprise a single concatemer bound thereto,

wherein said concatemers do not bind to said inter-regional areas, and

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

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

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

17. The method of claim 16, wherein said attractive noncovalent interactions are a member selected from Van der Waal forces, hydrogen bonding, and ionic interactions.

18. The method of claim 16, wherein said discrete spaced apart regions comprise functional moieties.

19. The method of claim 18, wherein said functional moieties comprise amines.

20. The method of claim 16 wherein the adaptor comprises a Type IIs endonuclease restriction site.

21. The method of claim 20, wherein said concatemers are generated from a circular template, wherein said circular template comprises said adaptor and said first target sequence.

22. The method of claim 20, wherein said target polynucleotide is a genomic sequence.

23. The method of claim 20, wherein substantially all of said plurality of discrete spaced apart regions have at most a single concatemer attached.

24. The method of claim 16 wherein more than 80% of said discrete regions have a single concatemer attached thereto.

25. The method of claim 16 wherein each concatemer comprises 100-1000 monomeric units.

29. A method for making an array of randomly disposed concatemers, wherein said method comprises:

(a) providing a composition comprising at least one million DNA concatemers, wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises;

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

(ii) an adaptor;

(b) providing a solid substrate comprising a surface and a plurality of discrete spaced apart regions defined on said surface in a regular array,

wherein the discrete regions are positively charged so as to bind said concatemers, and

wherein said discrete spaced apart regions are separated by hydrophobic inter-regional areas and are arranged on the surface in a regular array;

(c) depositing said composition on said surface, thereby randomly disposing concatemers on said plurality of discrete spaced apart regions such that at least 80% of said plurality of discrete spaced apart regions comprise a single concatemer bound thereto and said concatemers do not bind to said inter-regional areas.