NUCLEIC ACID AMPLIFICATION

• US 20130281307A1
• Filed: 06/20/2013
• Published: 10/24/2013
• 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
• Granted Patent: US 9,371,557 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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

1-20. -20. (canceled)

21. A method for nucleic acid amplification, comprising:

(a) forming a reaction mixture including a continuous liquid phase containing a plurality of supports, a plurality of different polynucleotide templates, a recombinase and a polymerase; and

(b) clonally amplifying, within the continuous liquid phase, at least two of the polynucleotide templates onto different supports under isothermal amplification conditions.

22. The method of claim 21, wherein at least two of the supports include a primer having a sequence that is complementary to a sequence within a polynucleotide template of the reaction mixture.

23. The method of claim 21, wherein at least two of the supports each include no more than one type of primer.

24. The method of claim 21, wherein the reaction mixture includes at least one primer in solution.

25. The method of claim 21, wherein the liquid phase of the reaction mixture includes a cellulose polymer.

26. The method of claim 21, wherein the reaction mixture includes at least 100 different polynucleotide templates and 100 separate supports.

27. The method of claim 21, wherein the recombinase includes a mutant, variant or recombinant uvsX protein.

28. The method of claim 21, further including forming a plurality of monoclonal amplicons during the clonally amplifying.

29. The method of claim 28, further including forming an array of monoclonal polynucleotide populations by distributing at least some of the monoclonal amplicons onto a surface.

30. The method of claim 28, wherein the surface includes a plurality of reaction sites, and the distributing includes distributing a single monoclonal amplicon to at least one of the reaction sites.

31. The method of claim 29, further including sequencing at least a portion of one or more of the monoclonal amplicons.

32. The method of claim 31, wherein the sequencing includes contacting one or more of the monoclonal amplicons with a sequencing reaction mixture including a polymerase.

33. The method of claim 32, wherein the sequencing reaction mixture includes a primer having a primer sequence that is complementary to a sequence within one or more monoclonal amplicons.

34. The method of claim 30, further including incorporating a nucleotide into at least one of the monoclonal amplicons.

35. The method of claim 30, wherein at least one of the reaction sites includes, or is linked to, a sensor capable of detecting a signal indicating the incorporating.

36. The method of claim 35, wherein the sensor includes a field effect transistor (FET).

37. The method of claim 36, wherein the signal includes an increase in concentration of hydrogen, phosphate or pyrophosphate in the sequencing reaction mixture.

38. The method of claim 21, wherein the clonally amplifying is performed without contacting the polynucleotide templates with a chemical denaturant during the clonally amplifying.