Methods of reducing density-dependent GC bias in amplification

• US 10,392,655 B2
• Filed: 06/02/2015
• Issued: 08/27/2019
• Est. Priority Date: 06/02/2014
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Boutell, Jonathan Mark, Shanahan, Susan, Rigatti, Roberto
• Primary Examiner(s): Bertagna, Angela M.
• Application Number:
  US15/315,342
• Publication Number:
  US 20180187251A1
• Time in Patent Office: 1,547 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 1/6846 Common amplification features
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• 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 invention provides methods for amplifying nucleic acids, particularly methods for reducing density-dependent GC bias and for reducing nucleic acid damage in a bridge amplification of a nucleic acid template. The invention also provides methods for evaluating the effect of reagents and/or additives on nucleic acid damage during bridge amplification of nucleic acid template strands. The methods are suited to solid phase amplification, for example, utilizing flow cells.

Claims

1. A method for reducing density-dependent GC bias and/or nucleic acid damage in bridge amplification of a double-stranded DNA template on a surface comprising:

(a) denaturing the double-stranded DNA template with a first solution comprising formamide and at least one type of dNTP to produce single-stranded DNA template strands;

(b) annealing the single-stranded DNA template strands to oligonucleotide primers bound to the surface; and

(c) extending the oligonucleotide primers by replacing the first solution with a solution comprising a polymerase and a mixture of different dNTPs, whereby the oligonucleotide primers bound to the surface are fully extended; and

(d) repeating steps (a)-(c) at least once;

wherein density-dependent GC bias and/or nucleic acid damage in the amplification of the double-stranded DNA template is reduced.

2. The method of claim 1, wherein first solution comprises at least one additive.

3. The method of claim 2, wherein the additive comprises a chelating agent.

4. The method of claim 2, wherein the additive comprises at least one citrate.

5. The method of claim 4, wherein the at least one citrate is selected from:

monosodium citrate, disodium citrate, trisodium citrate, and potassium citrate.

6. The method of claim 2, wherein the additive comprises EDTA.

7. The method of claim 2, wherein the additive comprises betaine.

8. The method of claim 2, wherein the additive comprises DMSO.

9. The method of claim 1, wherein a cluster of identical DNA strands is generated on the surface.

10. The method of claim 1, wherein the first solution is replaced by a second solution after step (a) and before step (b), wherein the second solution comprises water and/or a pre-mix solution comprising one or more components selected from betaine, Tris, ammonium sulfate, magnesium sulfate, polyethylene glycol tert-octylphenyl ether, and DMSO.

11. The method of claim 10, wherein the second solution comprises less than about 100 mM of salt.

12. The method of claim 11, wherein the second solution is substantially free from salt.

13. The method of claim 1, wherein the solution comprising the polymerase comprises less than about 100 mM of salt.

14. The method of claim 13, wherein the solution comprising the polymerase is substantially free from any salt.