Direct capture, amplification and sequencing of target DNA using immobilized primers

Patent metadata

Field	Value
Publication number	US-9309556-B2
Application number	US-201113239226-A
Country	US
Kind code	B2
Filing date	Sep 21, 2011
Priority date	Sep 24, 2010
Publication date	Apr 12, 2016
Grant date	Apr 12, 2016

Abstract

Certain embodiments provide a method for capturing a genomic fragment. The method may comprise: obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides; hybridizing a first member of the first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with the first member and a region that contains a genomic sequence; extending the first member of the first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to the genomic sequence; hybridizing the support-bound selection primer to a nucleic acid fragment comprising the genomic sequence; extending the support-bound selection primer to produce an extension product that contains a sequence that flanks the genomic sequence, e.g., in a genome; and amplifying the extension product on the substrate.

First claim

What is claimed is:

1. A method of capturing and amplifying a selected sequence comprising:
a) obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides, wherein the members of said first and second populations of surface-bound oligonucleotides are attached to the same substrate but not spatially addressed on said substrate;
b) hybridizing a first member of said first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with said first member and a region that contains a genomic sequence,
c) extending said first member of said first population of surface-bound oligonucleotides using the hybridized selection oligonucleotide of (b) as a template to produce an extended support-bound selection primer that comprises a sequence that is complementary to said genomic sequence;
d) hybridizing said extended support-bound selection primer of step c) to denatured fragmented genomic DNA to produce a duplex comprising said extended support-bound selection primer and a strand of genomic DNA that comprises said genomic sequence, wherein, in the duplex, the strand of genomic DNA is at least 100 bases in length and the 3′ end of the extended support-bound selection primer is extendible using the strand of genomic DNA as a template;
e) extending said extended support-bound selection primer using the strand of genomic DNA as a template to produce an extension product that contains the complement of a sequence that flanks said genomic sequence; and
f) amplifying said extension product of step e) on said substrate by bridge PCR using unextended members of said first and second populations of surface-bound oligonucleotides of step a), to produce a PCR product.

2. The method of claim 1 , wherein said strand of genomic DNA is comprises a 5′ end adaptor, wherein said extending of step e) produces an extension product that comprises, on its 3′ end, a sequence that is complementary to said adaptor, and wherein members of said second population of said surface-bound oligonucleotides hybridize to said sequence that is complementary to said adaptor during said amplifying step f).

3. The method of claim 2 , wherein said 5′ end adaptor comprises a binding site for a sequencing primer.

4. The method of claim 1 , wherein said method comprises, between steps e) and f), ligating an adaptor onto the 3′ end of said extension product, and wherein members of said second population of said surface-bound oligonucleotides hybridize to said adaptor during said amplifying.

5. The method of claim 4 , wherein said adaptor comprises a binding site for a sequencing primer at the end that is ligated to said extension product.

6. The method of claim 1 , wherein said second population of surface-bound oligonucleotides are made by:
i. hybridizing members of an initial second population of surface-bound oligonucleotides to an oligonucleotide comprising a region that hybridizes with the members of said second population of surface-bound oligonucleotides and a region that is complementary to a sequence of said strand of genomic DNA; and
ii. extending said members of said initial second population of surface-bound oligonucleotides to produce said second population of surface-bound oligonucleotides.

7. The method of claim 1 , wherein said selection oligonucleotide comprises a binding site for a sequencing primer between said region that hybridizes with said first member and said region that contains said genomic sequence.

8. The method of claim 1 , further comprising sequencing a first strand of said PCR product to obtain at least part of the nucleotide sequence of said sequence that flanks said genomic sequence, or complement thereof.

9. The method of claim 1 , further comprising sequencing the second strand of said PCR product to obtain at least part of the nucleotide sequence of said sequence that flanks said genomic sequence, or complement thereof.

10. The method of claim 1 , wherein said method comprises fragmenting a mammalian genome to produce a fragmented genome, optionally adding adaptors to said fragmented genome, and applying said fragmented genome to said substrate.

11. The method of claim 10 , wherein said fragmenting is done physically, chemically or using a restriction enzyme.

12. The method of claim 11 , wherein said fragmenting is done by sonication or shearing.

13. The method of claim 10 , wherein said hybridizing is done by preparing a plurality of fragmented genomes from a plurality of different individuals, pooling said plurality of fragmented genomes to produce a pool, applying said pool of fragmented genomes to said substrate, and obtaining PCR products that comprise a sequence that flanks said genomic sequence in said different individuals.

14. The method of claim 13 , further comprising sequencing at least the first strand of said PCR products to obtain at least part of the nucleotide sequence of said sequence that flanks said genomic sequence in said different individuals.

15. The method of claim 14 , wherein, prior to pooling, different adaptors are ligated to said fragmented genomes from said different individuals, wherein said the adaptor comprises a barcode sequence that allows the source of a sequence to be identified.

16. The method of claim 15 , wherein said method comprises:
adaptor-ligating fragmented genomic DNA from a first subject using a first adaptor that comprises a first barcode sequence to produce a first product;
adaptor-ligating fragmented genomic DNA from a second subject using a second adaptor that comprises a second barcode sequence to produce a second product;
combining said first and second products to produce a mixed template; and
performing the method of claim 1 using said mixed template to provide first and second PCR product each containing said barcode sequence.

17. The method of claim 16 , wherein said mixed template comprises fragmented genomic DNA from at least 1,000 subjects.

18. The method of claim 1 , comprising:
i. ligating the fragmented genomic DNA to an adaptor that contains a site for a sequencing primer and a nucleotide sequence that is the same as the second surface bound oligonucleotides,
ii. hybridizing the adaptor-ligated fragments to a first member of the first population of surface-bound oligonucleotides,
iii. extending the first member of the first population of surface-bound oligonucleotides to which the adaptor ligated fragment is hybridized; and
iv. hybridizing the adaptor-containing end of the extension product to a second support bound polynucleotide, thereby producing a bridge and facilitating bridge PCR.

19. The method of claim 1 , wherein said second population of surface-bound oligonucleotides are made by ligating an oligonucleotide comprising a region that is complementary to a sequence of said strand of genomic DNA to an initial second population of surface-bound oligonucleotides to produce said second population of surface-bound oligonucleotides.

20. A method of capturing and amplifying a selected sequence comprising the following steps, in order:
a) obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides, wherein the members of said first and second populations of surface-bound oligonucleotides are attached to the same substrate but not spatially addressed on said substrate;
b) hybridizing a first member of said first population of surface-bound ol

Assignees

• Myllykangas Samuel
• Buenrostro Jason
• Ji Hanlee P
• Univ Leland Stanford Junior

Inventors

• Myllykangas Samuel
• Buenrostro Jason
• Ji Hanlee P

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title