Nucleic acid amplification

• US 9,309,557 B2
• Filed: 03/15/2013
• Issued: 04/12/2016
• 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, Chen, Shiaw-Min, O'Neil, Jennifer, Kasinskas, Rachel, Rothberg, Jonathan, Li, Bin, Lao, Kai Qin
• Primary Examiner(s): Mummert, Stephanie K
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US13/842,296
• Publication Number:
  US 20130225421A1
• Time in Patent Office: 1,124 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 within a continuous liquid phase under nucleic acid synthesis conditions by distributing at least two different polynucleotide templates comprising both a first primer binding sequence and a second primer binding sequence, into an array of reaction chambers, wherein the array or reaction chambers is on a support and contains a first universal primer that does not include any target-specific sequence, by introducing a polymerase, a second universal primer in solution, and said at least two different polynucleotide templates into different reaction chambers of the array, 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;

B. forming at least two substantially monoclonal nucleic acid populations by using the polymerase to amplify under isothermal conditions a single one of each of said at least two different polynucleotide templates within said different reaction chambers, by partially denaturing and without first compartmentalizing, within the same reaction mixture of step (a) in the continuous liquid phase, wherein the different reaction chambers are in fluid communication with each other during the amplifying;

C. after the amplifying, distributing a plurality of additional polynucleotide templates into the array of reaction chambers; and

D. amplifying under the isothermal conditions, at least one polynucleotide template of the plurality of additional polynucleotide templates, thereby forming an additional substantially monoclonal nucleic acid population within each of at least one additional reaction chamber of the array of reaction chambers that does not include any other substantially monoclonal nucleic acid population.

2. The method of claim 1, wherein the amplifying includes:

contacting the at least two different polynucleotide templates with a recombinase in the reaction chambers.

3. The method of claim 1, wherein at least one of the reaction chambers is operatively coupled to a sensor.

4. The method of claim 3, wherein the sensor can detect the presence of a nucleotide incorporation byproduct within the at least one reaction chamber.

5. The method of claim 3, wherein the at least one reaction chamber includes a hydrophilic polymer matrix conformally disposed within the at least one reaction chamber.

6. The method of claim 5, wherein the hydrophilic polymer matrix includes a hydrogel polymer matrix.

7. The method of claim 5, wherein the hydrophilic polymer matrix is a cured-in-place polymer matrix.

8. The method of claim 5, wherein the hydrophilic polymer matrix includes polyacrylamide, copolymers thereof, derivatives thereof, or combinations thereof.

9. The method of claim 8, wherein the polyacrylamide is conjugated with an oligonucleotide primer.

10. The method of claim 5, wherein the at least one reaction chamber has a diameter in a range of 0.1 micrometers to 2 micrometers.

11. The method of claim 3, wherein the sensor includes a field effect transistor (FET).

12. The method of claim 11, further including detecting the presence of one or more nucleotide incorporation byproducts within the at least one reaction chamber of the array using the FET.

13. The method of claim 11, further including detecting a pH change occurring within the at least one reaction chamber using the FET.

14. The method of claim 11, further including introducing a nucleotide into the at least one reaction chamber;

and detecting an output signal from the sensor resulting from incorporation of the nucleotide into a primer annealed to a polynucleotide within the at least one reaction chamber.

15. The method of claim 14, wherein the output signal is based on a threshold voltage of the FET.

16. The method of claim 11, wherein the FET includes a floating gate conductor coupled to the at least one reaction chamber.

17. The method of claim 11, wherein the FET includes a floating gate structure comprising a plurality of conductors electrically coupled to one another and separated by dielectric layers, and the floating gate conductor is an uppermost conductor in the plurality of conductors.

18. The method of claim 11, wherein the FET includes an ion sensitive FET (ISFET).

19. The method of claim 1, further comprising successfully performing a genetic sequencing technique on 40% or more of the reaction chambers of the array of reaction chambers.

20. The method of claim 1, further comprising successfully performing a genetic sequencing technique on 50% or more of the reaction chambers of the array of reaction chambers.

21. The method of claim 1, wherein the amplifying the at least one polynucleotide template of the plurality of additional polynucleotide templates, is performed by partially denaturing and without first compartmentalizing, within the same reaction mixture of step (a) in the continuous liquid phase, wherein the different reaction chambers are in fluid communication with each other during the amplifying the at least one polynucleotide template of the plurality of additional polynucleotide templates.