Polynucleotide modification on solid support

• US 9,677,132 B2
• Filed: 12/18/2014
• Issued: 06/13/2017
• Est. Priority Date: 01/16/2014
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Incorporated
• Inventors: Boutell, Jonathan Mark, Rigatti, Roberto, Gormley, Niall Anthony, Eckhardt, Allen E.
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/575,863
• Publication Number:
  US 20150197799A1
• Time in Patent Office: 908 Days
• Field of Search: None
• 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 1/6853 using modified primers or templates
• 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 2521/507 Recombinase
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 present disclosure relates to the field of molecular biology and more specifically to methods for capturing and amplifying target polynucleotides on a solid surface.

Claims

1. A method of modifying an immobilized capture primer comprising:

a. providing a solid support having an immobilized application-specific capture primer, the application-specific capture primer comprising;

i. a 3′

portion comprising an application-specific capture region, and

ii. a 5′

portion comprising a universal capture region;

b. contacting an application-specific polynucleotide with the application-specific capture primer under conditions sufficient for hybridization to produce an immobilized application-specific polynucleotide, and

c. removing the application-specific capture region of an application-specific capture primer not hybridized to an application-specific polynucleotide to convert the unhybridized application-specific capture primer to a universal capture primer.

2. The method of claim 1, wherein the application-specific capture primer comprises a plurality of different immobilized application-specific capture primers.

3. The method of claim 1, wherein the application-specific polynucleotide comprises a plurality of different application-specific polynucleotides.

4. The method of claim 1, wherein a portion of the application-specific capture region is removed.

5. The method of claim 1, wherein the application-specific capture region comprises a target-specific capture region and wherein the application-specific polynucleotide comprises a target polynucleotide.

6. The method of claim 5, further comprising extending the target-specific capture region of an application-specific capture primer hybridized to a target polynucleotide to produce an immobilized extension product complementary to the target polynucleotide.

7. The method of claim 6, further comprising annealing the universal capture primer to the immobilized extension product.

8. The method of claim 7, further comprising amplifying by PCR the immobilized extension product to produce a plurality of immobilized amplicons.

9. The method of claim 8, further comprising sequencing the plurality of immobilized amplicons.

10. The method of claim 1, wherein the application-specific capture region comprises a transposon end (TE) region and wherein the application-specific polynucleotide comprises a TE oligonucleotide.

11. The method of claim 10, further comprising binding a transposase to the TE region—

TE oligonucleotide hybrid after execution of step b) and prior to execution of step c) to produce a support bound transposome complex.

12. The method of claim 11, further comprising contacting the support bound transposome complex with a target polynucleotide under conditions wherein the support-bound transposome complex joins the 3′

-end of the TE region in the application-specific capture primer (the “

transferred strand”

) to the target polynucleotide to produce an immobilized target polynucleotide.

13. The method of claim 1, wherein the universal capture region comprises a known sequence.

14. The method of claim 1, further comprising applying an oligonucleotide before execution of step c) under conditions sufficient for the oligonucleotide to hybridize with the universal capture region of an application-specific capture primer to produce a double-stranded DNA region.

15. The method of claim 1, further comprising contacting the application-specific capture primer with a nuclease, wherein the application-specific capture region of an application-specific capture primer not hybridized with an application-specific polynucleotide is removed by the nuclease.

16. The method of claim 15, wherein the nuclease is an exonuclease.

17. A method of modifying an immobilized capture primer comprising:

a. providing a solid support having an immobilized application-specific capture primer, said application-specific capture primer comprising;

i. a 3′

portion comprising a target-specific capture region, and

ii. a 5′

portion comprising a universal capture region;

b. contacting a target polynucleotide with the application-specific capture primer under conditions sufficient for hybridization to produce an immobilized target-specific polynucleotide;

c. extending the application-specific capture primer hybridized to the immobilized target-specific polynucleotide to produce an immobilized extension product complementary to the immobilized target-specific polynucleotide;

d. applying an oligonucleotide under conditions sufficient for the oligonucleotide to hybridize with the universal capture region of the immobilized application-specific capture primer;

e. contacting the immobilized application-specific capture primer with nuclease under conditions sufficient for the nuclease to remove the target-specific capture region of an application-specific capture primer not hybridized to the immobilized target-specific polynucleotide to convert the unhybridized application-specific capture primer to a universal capture primer;

f. removing the oligonucleotide from the universal capture primer;

g. annealing the universal capture primer to the immobilized extension product;

h. amplifying the immobilized extension product to produce a plurality of immobilized amplicons, wherein amplifying comprises a bridge amplification step, and

i. sequencing the plurality of immobilized amplicons.

18. The method of claim 17, wherein the nuclease is an exonuclease.