Efficient arrays of amplified polynucleotides

• US 7,910,354 B2
• Filed: 10/29/2007
• Issued: 03/22/2011
• Est. Priority Date: 10/27/2006
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Callow, Matthew, Drmanac, Radoje T.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Thomas; David C
• Application Number:
  US11/927,356
• Publication Number:
  US 20080318796A1
• Time in Patent Office: 1,240 Days
• Field of Search: None
• US Class Current: 435/287.2
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are arrays of polynucleotides in which the polynucleotides have undergone multiple rounds of amplification in order to increase the strength of signals associated with single polynucleotide molecules.

Claims

1. A random array of polynucleotides, wherein said random array comprises:

a) a substrate having a surface; and

b) a plurality of concatemer extension products disposed on said surface, wherein;

i) said plurality of concatemer extension products is formed from in situ amplification of immobilized concatemers, and

ii) each of said immobilized concatemers comprises multiple copies of a monomer, and said monomer comprises a first interspersed adaptor that is located between a first and second target sequence of a target polynucleotide, wherein said first and second target sequences are contiguous segments of said target polynucleotide.

2. The random array of claim 1, wherein:

a) said surface comprises capture probes, and

b) said plurality of concatemer extension products are immobilized to said surface through duplexes formed between said concatemer extension products and said capture probes.

3. The random array of claim 2, wherein said capture probes are distributed in discrete regions on said surface, and wherein areas surrounding said discrete regions do not bind said immobilized concatemers or said concatemer extension products.

8. The random array of claim 1 or 4, wherein for a number of said immobilized concatemers, different concatemers comprise first and second target sequences from different target polynucleotides.

4. A random array of target polynucleotides of unknown sequence, wherein said random array comprises a substrate having a surface and a plurality of concatemer extension products disposed on said surface, wherein:

(a) said plurality of concatemer extension products is formed from in situ amplification of immobilized concatemers, and

(b) said immobilized concatemers are formed from rolling circle replication of a circular template that comprises;

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

(ii) a first interspersed adaptor;

(iii) a second target sequence of said target polynucleotide, wherein said first and second target sequences are contiguous segments of said target polynucleotide and said first interspersed adaptor is located between said first and second target sequence.

5. The random array of claim 4, wherein:

a) said surface comprises capture probes, and

b) said plurality of concatemer extension products are immobilized to said surface through duplexes formed between said concatemer extension products and said capture probes.

6. The random array of claim 4, wherein said capture probes are distributed in discrete regions on said surface, and wherein areas surrounding said discrete regions do not bind said concatemers or said concatemer extension products.

7. The random array of claim 4, wherein at least a portion of said first or second target sequences have been identified in a sequencing reaction.