Single Molecule Arrays for Genetic and Chemical Analysis

• US 20110071053A1
• Filed: 09/15/2010
• Published: 03/24/2011
• Est. Priority Date: 06/15/2005
• Status: Abandoned Application

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Yeung, George, Drmanac, Snezana, DRMANAC, Radoje, Callow, Matthew J., Hauser, Brian K.
• Application Number:
  US12/882,880
• Publication Number:
  US 20110071053A1
• Time in Patent Office: Days
• US Class Current: 506/17
• 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 and, wherein each of said discrete regions has an area;

(b) a population of concatemers randomly disposed on said plurality of discrete regions such that at least a majority of said plurality of discrete regions comprises a single concatemer attached thereto and said inter-regional areas are substantially free of concatemers, wherein said concatemers are generated from a circular template and comprise a plurality of monomeric units, and wherein each monomeric unit comprises;

(i) a first target sequence of a target polynucleotide, and

(ii) an adaptor,

and wherein said concatemers have a diameter that is approximately equal to that of said discrete spaced apart regions,

and wherein said population of concatemers comprises a plurality of different target polynucleotides.

2. The random array of claim 1, wherein said target polynucleotide is genomic DNA.

3. The random array of claim 2, wherein said genomic DNA is human genomic DNA.

4. The random array of claim 1, wherein said concatemers are non-covalently immobilized to said discrete regions.

5. The random array of claim 1, wherein said substrate comprises silica.

6. The random array of claim 1, wherein said discrete regions are arranged on said surface in a pattern.

7. The random array of claim 1, wherein said circular template comprises said adaptor and said first target sequence.

8. The random array of claim 1, wherein said discrete regions have a nearest neighbor distance of 200 nm or greater.

9. The random array of claim 1, wherein said discrete regions comprise an amino group.

10. The random array of claim 1, wherein said area of said discrete spaced apart regions is less than 1 μ

m².

11. The random array of claim 1, wherein said area of said discrete spaced apart regions is in the range of from 0.04 μ

m² to 1 μ

m².

12. The random array of claim 1, wherein a majority of said concatemers have a size in the range of 50-250 kb.

13. The random array of claim 1, wherein said discrete regions are prepared by photolithography.

14. 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, wherein each of said discrete regions has an area;

(b) a population of concatemers randomly disposed on said plurality of discrete regions such that at least a majority of said plurality of discrete regions comprises a single concatemer attached thereto, wherein said concatemers are generated from a circular template, wherein said inter-regional areas are substantially free of concatemers, and wherein said concatemers comprise a plurality of monomeric units and each monomeric unit comprises;

(i) a first target sequence of a target polynucleotide, and

(ii) an adaptor,

wherein each of said discrete regions comprise a number of reactive functionalities, and

wherein said number is such that a single concatemer occupies substantially all of said number of reactive functionalities, and

wherein said population of concatemers comprises a plurality of different target polynucleotides.

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 areas, wherein each of said discrete regions has an area;

(b) a population of concatemers randomly disposed on said plurality of discrete regions, wherein each of said discrete regions has an area corresponding in size to that of said concatemers such that at least a majority of said plurality of discrete regions comprises a single concatemer attached thereto, wherein said inter-regional areas are substantially free of concatemers, and wherein said concatemers comprise a plurality of monomeric units and each monomeric unit comprises;

(i) a first target sequence of a target polynucleotide, and

(ii) an adaptor,

wherein said population of concatemers comprises a plurality of different target polynucleotides.