Amplification methods to minimise sequence specific bias

• US 9,469,872 B2
• Filed: 06/09/2014
• Issued: 10/18/2016
• Est. Priority Date: 02/23/2010
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Rigatti, Roberto, Boutell, Jonathan Mark, Shen, Min-Jui Richard
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/299,269
• Publication Number:
  US 20140329246A1
• Time in Patent Office: 862 Days
• Field of Search: 435/91.2
• US Class Current: 1/1
• 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) a first round comprising one or more cycles of amplification, wherein the cycles of amplification comprise amplifying nucleic acid templates in an ensemble under conditions favoring copying AT rich templates over GC rich templates such that the efficiency of amplification of AT rich templates is not reduced or inhibited relative to non-AT rich templates;

and

(b) a second round comprising one or more cycles of amplification, wherein the cycles of amplification comprise amplifying the nucleic acid templates in the ensemble under conditions favoring copying GC rich templates over AT rich templates such that the efficiency of amplification of AT rich templates is not reduced or inhibited relative to non-AT rich templates;

wherein the nucleic acid templates are immobilized on a solid support, and

wherein steps (a) and (b) are repeated one or more times.

2. The method of claim 1, wherein the first round comprises one to fifty cycles of amplification and the second round comprises one to fifty cycles of amplification.

3. The method of claim 1, wherein the first round comprises at least five cycles of amplification and the second round comprises at least fifteen cycles of amplification.

4. The method of claim 1, wherein the concentration of dATP or dTTP in step (b) is less than the concentration of dGTP and dCTP in step (a).

5. The method of claim 1, wherein the concentration of dATP or dTTP is less one tenth the concentration of dGTP and dCTP.

6. The method of claim 1, wherein the concentration of dATP or dTTP in step (b) is less than 20 micromolar.

7. The method of claim 1, wherein one or more additives are used in step (b) but not in step (a).

8. The method of claim 7, wherein the additive is ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-dGTP, acetamide, betaine or tetramethylammonium chloride (TMACL).

9. The method of claim 8, wherein the betaine is present at a concentration selected from the group consisting of 2.5, 3, 3.25, 3.5, 3.75 or 4M.

10. The method of claim 8, wherein the betaine is present at a concentration between about 1 M and about 5 M.

11. The method of claim 8, wherein a combination of betaine and DMSO is used.

12. The method of claim 8, wherein betaine, DMSO and 7-deaza-dGTP are used.

13. The method of claim 1, wherein the annealing temperature in step (b) is higher than the annealing temperature in step (a).

14. The method of claim 13, wherein the annealing temperature in step (b) is 55−

65°

C.

15. The method of claim 13, wherein the annealing temperature in step (b) is 58−

62°

C.

16. The method of claim 1, wherein the concentration of salt in step (b) is lower than the concentration of salt in step (a).

17. The method of claim 1, wherein the pH in step (b) is higher than the pH in step (a).

18. The method of claim 1, wherein the ionic strength in step (b) is lower than the ionic strength in step (a).

19. The method of claim 1, wherein step (b) occurs prior to step (a).

20. The method of claim 1, wherein the solid support is selected from the group consisting of a resin, gel, bead, well, column, chip, membrane, matrix, plate, and filter.