Nucleic acid amplification

• US 9,371,557 B2
• Filed: 06/20/2013
• Issued: 06/21/2016
• Est. Priority Date: 12/17/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Li, Chieh-Yuan, Ruff, David, O'Neil, Jennifer, Kasinskas, Rachel, Chen, Shiaw-Min, Rothberg, Jonathan, Li, Bin, Lao, Kai Qin
• Primary Examiner(s): Mummert, Stephanie K
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US13/923,232
• Publication Number:
  US 20130281307A1
• Time in Patent Office: 1,097 Days
• Field of Search: None
• US Class Current: 1/1
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

In some embodiments, the present teachings provide methods for nucleic acid amplification, comprising forming a reaction mixture, and subjecting the reaction mixture to conditions suitable for nucleic acid amplification. In some embodiments, methods for nucleic acid amplification include subjecting the nucleic acid to be amplified to partially denaturing conditions. In some embodiments, methods for nucleic acid amplification include amplifying without fully denaturing the nucleic acid that is amplified. In some embodiments, the methods for nucleic acid amplification employ an enzyme that catalyzes homologous recombination and a polymerase. In some embodiments, methods for nucleic acid amplification can be conducted in a single reaction vessel. In some embodiments, methods for nucleic acid amplification can be conducted in a single continuous liquid phase of a reaction mixture, without need for compartmentalization of the reaction mixture or immobilization of reaction components. In some embodiments, methods for nucleic acid amplification comprise a amplifying at least one polynucleotide onto a surface under isothermal amplification conditions, optionally in the presence of a polymer. The polymer can include a sieving agent and/or a diffusion-reducing agent.

Claims

1. A method for nucleic acid amplification, comprising:

(a) forming a reaction mixture including a continuous liquid phase containing (i) a plurality of separate supports containing a first universal primer that does not include any target-specific sequence, (ii) a second universal primer in solution, (iii) a plurality of different polynucleotide templates containing both a first primer binding sequence and a second primer binding sequence, wherein the first primer binding sequence is complementary or identical to at least a portion of the first universal primer and the second primer binding sequence is complementary or identical to at least a portion of the second universal primer, and (iv) a polymerase; and

(b) forming two or more substantially monoclonal populations, each linked to a different support, by clonally amplifying, within the same reaction mixture of step (a) in the continuous liquid phase, at least two of the polynucleotide templates on different separate supports under isothermal amplification conditions using the first and second universal primers by partially denaturing and without compartmentalizing the polynucleotide templates.

2. The method of claim 1, wherein at least two of the supports each include only one type of first universal primer.

3. The method of claim 1, wherein the liquid phase of the reaction mixture includes a dissolved cellulose polymer.

4. The method of claim 1, wherein the reaction mixture includes at least 100 different polynucleotide templates and 100 separate supports.

5. The method of claim 1, further including forming an array of substantially monoclonal amplicons by distributing at least some of the different supports, each attached to a substantially monoclonal population, onto a surface.

6. The method of claim 5, further including sequencing at least a portion of one or more substantially monoclonal populations.

7. The method of claim 6, wherein the sequencing includes contacting one or more of the substantially monoclonal populations with a sequencing reaction mixture including a polymerase.

8. The method of claim 7, wherein the sequencing reaction mixture includes a primer having a primer sequence that is complementary to a sequence within one or more of the substantially monoclonal populations.

9. The method of claim 5, wherein the array includes, or is linked to, a sensor capable of detecting a signal indicating nucleotide incorporation and production of a nucleotide incorporation byproduct.

10. The method of claim 9, wherein the sensor includes a field effect transistor (FET).

11. The method of claim 10, wherein the signal includes an increase in concentration of hydrogen, phosphate or pyrophosphate in the sequencing reaction mixture.

12. The method of claim 1, wherein the clonally amplifying is performed without contacting the polynucleotide templates with a chemical denaturant during the clonally amplifying.

13. The method of claim 1, wherein the reaction mixture further includes a recombinase.

14. The method of claim 13, wherein the recombinase includes a mutant, variant or recombinant uvsX protein.

15. The method of claim 5, further including incorporating a nucleotide into one or more polynucleotides of at least one substantially monoclonal amplicon of the array, or into a primer hybridized to the one or more polynucleotides.