Amplification methods to minimise sequence specific bias

• US 10,428,363 B2
• Filed: 10/17/2016
• Issued: 10/01/2019
• Est. Priority Date: 02/23/2010
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Rigatti, Roberto, Boutell, Jonathan Mark, Shen, Min-Jui Richard
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US15/295,811
• Publication Number:
  US 20170029857A1
• Time in Patent Office: Days
• Field of Search: 435 912
• CPC Class Codes: C09K 3/00 Materials not provided for elsewhere
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Methods for amplifying nucleic acids are provided. The methods can be used to minimize sequence specific bias caused by the preferential amplification of certain nucleic acid sequences. Methods are described which can lower the efficiency of AT rich templates relative to GC rich templates, thereby minimizing GC bias during amplification reactions with multiple templates of different sequence. The methods are suited to solid phase amplification, for example, utilizing flow cells.

Claims

1. A method for amplifying nucleic acid templates of different sequences comprising:

a) amplifying nucleic acid templates on a solid support in the presence of an additive capable of increasing efficiency of amplification of GC rich nucleic acid templates or decreasing efficiency of amplification of AT rich nucleic acid templates, wherein a subset of nucleotide types are incorporated into copies of the templates at a lower efficiency compared to at least one other type of nucleotide, thereby producing a plurality of different copies; and

b) using the plurality of different copies as templates in a second cycle of amplification.

2. The method of claim 1, wherein the additive is betaine.

3. The method of claim 2, wherein the betaine is present at a concentration selected from the group consisting of 2.5, 3, 3.25, 3.5, 3.75, and 4 M.

4. The method of claim 2, wherein the betaine is present at a concentration between about 1 M and about 5 M.

5. The method of claim 1, wherein the method comprises at least one cycle of amplification using four nucleotide types which are not present at the same concentration.

6. The method of claim 5, wherein the amplification process also comprises at least one cycle using four nucleotide types which are present at the same concentration.

7. The method of claim 6, wherein the cycle using four nucleotide types which are present at the same concentration is followed by at least one cycle of amplification using four nucleotide types which are not present at the same concentration.

8. The method of claim 1, wherein the nucleotides are deoxynucleotide triphosphates selected from the group consisting of dATP, dTTP, dCTP, and dGTP.

9. The method of claim 8, wherein the concentration of dATP or dTTP is less than the concentrations of dGTP and dCTP.

10. The method of claim 9, wherein the concentration of dATP or dTTP is less than half, less than one quarter, or less than one tenth the concentration of dGTP and less than half the concentration of dCTP.

11. The method of claim 8, wherein the concentration of dATP or dTTP is less than 20 μ

M or less than 10 μ

M.

12. The method of claim 1, comprising the use of a T or A nucleotide analogue which makes the AT base pair interaction stronger.

13. The method of claim 1, comprising the use of a T or A nucleotide analogue which is incorporated by a polymerase with lower efficiency than dATP or dTTP.

14. The method of claim 13, wherein the T nucleotide analogue is 2-thio dTTP.

15. The method of claim 1, comprising the use of a T or A nucleotide analogue which causes a strand termination upon incorporation.

16. The method of claim 15, wherein the strand termination is reversible.

17. The method of claim 15, comprising the use of dUTP as a partial replacement for dTTP.

18. The method of claim 17, comprising the use of an archaeal polymerase.

19. The method of claim 18, wherein the archaeal polymerase is Pfu polymerase or a derivative thereof.