NUCLEIC ACID AMPLIFICATION

• US 20160032375A1
• Filed: 07/01/2015
• Published: 02/04/2016
• 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, KASINSKAS, Rachel, CHEN, Shiaw-Min, ROTHBERG, Jonathan, O'NEIL, Jennifer
• Granted Patent: US 10,113,195 B2
• Time in Patent Office: Days
• 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 by contacting, within a continuous liquid phase a first support, a second support, a first target polynucleotide template in solution, a second target polynucleotide template in solution, a first universal primer in solution that is optionally hybridized to at least one of the polynucleotide templates, and a polymerase, wherein the first and second target polynucleotide templates each include a first primer binding sequence and a second primer binding sequence; and

wherein the first and second supports each include the same second universal primer; and

wherein the first universal primer contains a sequence that is complementary or identical to the first primer-binding sequence; and

wherein the second universal primer contains a sequence that is complementary or identical to the second primer-binding sequence and does not include any target-specific sequence; and

(b) forming two or more substantially monoclonal populations, each linked to a different support, by clonally amplifying, without first compartmentalizing the first and second target polynucleotide templates, the first target polynucleotide template on the first support and the second target polynucleotide template on the second support, under isothermal amplification conditions.

2. The method of claim 1, wherein the continuous liquid phase includes a dissolved cellulose polymer.

3. The method of claim 1, wherein the continuous liquid phase includes at least 100 different polynucleotide templates and 100 separate supports.

4. The method of claim 1, further including forming an array of monoclonal polynucleotide populations by distributing at least some of the substantially monoclonal populations onto a surface.

5. The method of claim 1, further including sequencing a polynucleotide of one or more of the substantially monoclonal populations.

6. The method of claim 5, wherein the sequencing includes contacting the polynucleotide of the one or more of the substantially monoclonal populations with a sequencing reaction mixture including a polymerase.

7. The method of claim 6, wherein the sequencing reaction mixture includes a sequencing primer having a sequence that is complementary or identical to a corresponding sequence within the polynucleotide of the one or more substantially monoclonal populations.

8. The method of claim 7, further including incorporating a nucleotide into the sequencing primer.

9. The method of claim 8, further including detecting a signal indicating the incorporating using a sensor.

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

11. The method of claim 9, 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 12, wherein the chemical denaturant is selected from the group consisting of:

urea, formamide, NaOH and guanidine-containing agents.

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

15. The method of claim 14, wherein the recombinase includes a wild-type, mutant, variant or recombinant uvsX protein.

16. The method of claim 14, wherein the reaction mixture further includes an accessory factor for the uvsX protein.

17. The method of claim 16, wherein the reaction mixture further includes a wild-type, mutant, variant or recombinant uvsY protein.