Methods for selecting and amplifying polynucleotides

• US 8,999,642 B2
• Filed: 08/25/2009
• Issued: 04/07/2015
• Est. Priority Date: 03/10/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Sabot, Andrea, Rigatti, Roberto, Shen, Min-Jui Richard
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US13/387,078
• Publication Number:
  US 20120122737A1
• Time in Patent Office: 2,051 Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The invention provides methods for controlling the density of different molecular species on the surface of a solid support. A first mixture of different molecular species is attached to a solid support under conditions to attach each species at a desired density, thereby producing a derivatized support having attached capture molecules. The derivatized support is treated with a second mixture of different molecular species, wherein different molecular species in the second mixture bind specifically to the different capture molecules attached to the solid support. One or more of the capture molecules can be reversibly modified such that the capture molecules have a different activity before and after the second mixture of molecular species are attached. In particular embodiments, the different molecular species are nucleic acids that are reversibly modified to have different activity in an amplification reaction.

Claims

1. A method of selecting and amplifying polynucleotides on a solid support, comprising:

a) providing a plurality of amplification oligonucleotides immobilized on a solid support;

b) hybridizing a population of oligonucleotide probes to a subset of said amplification oligonucleotides, each of said oligonucleotide probes comprising a first portion which is complementary to the amplification oligonucleotides and a second portion which comprises sequence from a selected region of a template polynucleotide;

c) performing an extension reaction to extend hybridised amplification oligonucleotides to produce a population of support-bound capture oligonucleotides and a population of unextended amplification oligonucleotides, each capture oligonucleotide in said population comprising a sequence that is complementary to a selected region of a template polynucleotide;

d) applying a population of template polynucleotides to the solid support under conditions such that the template polynucleotides selectively hybridise to the support-bound capture oligonucleotides;

e) extending the support-bound capture oligonucleotides that are hybridized to the template polynucleotides, thereby generating extension products, the extension products having a complementary to the template polynucleotides and a portion complementary to the unextended amplification oligonucleotides; and

f) amplifying the extension products, wherein the amplifying comprises annealing one or more of the unextended amplification oligonucleotides to one or more of the extension products, thereby producing a solid-phase amplification product.

2. The method of claim 1, further comprising:

g) sequencing a first strand of the solid-phase amplification product of step f) to obtain at least part of the nucleotide sequence of the template polynucleotide.

3. The method of claim 2, wherein said solid-phase amplification products comprise a binding site for a universal sequencing primer.

4. The method of claim 1 wherein the capture oligonucleotides comprise at least 10 different capture sequences.

5. The method of claim 1 wherein the amplification oligonucleotides are reversibly blocked during the extension of the capture oligonucleotides.

6. The method of claim 5 wherein reversible blocking is by a chemical species attached to a 3′

end of the amplification oligonucleotides.

7. The method of claim 6 wherein the chemical species is a phosphate group.

8. The method of claim 1 wherein the amplification is isothermal.

9. The method of claim 1 wherein a portion complementary to the unextended amplification oligonucleotides comprises an adapter sequence.

10. The method of claim 9, wherein the template polynucleotides in the nucleic acid sample comprise different sequences and the adapter sequence is the same for each template polynucleotides.

11. The method of claim 10, wherein the plurality of amplification oligonucleotides each comprise a common sequence that is complementary to the adapter sequence that is the same for each template polynucleotides.

12. The method of claim 1, wherein the template polynucleotides comprise PCR amplicons.

13. The method of claim 1, wherein the template polynucleotides comprise genomic DNA fragments.

14. The method of claim 1, wherein the template polynucleotides are derived from cell-free DNA.

15. The method of claim 1, wherein the method further comprises the step of subjecting the extension products of step (e) to denaturation conditions prior to step (f).

16. The method of claim 1, wherein the method further comprises a wash step prior to step (f).