Nucleic acid amplification

• US 9,334,531 B2
• Filed: 09/10/2013
• Issued: 05/10/2016
• 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): PRIEST, AARON A
• Application Number:
  US14/023,361
• Publication Number:
  US 20140080717A1
• Time in Patent Office: 973 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• 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 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

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) distributing at least two different polynucleotide templates onto an array of reaction sites on a support wherein the array of reaction sites comprise the same first universal primer that does not include a template sequence by introducing a single one of each of said different polynucleotide templates into at least two different reaction sites of the array, wherein the polynucleotide templates each include the same first universal primer binding site, the same second universal primer binding site, and are present within a reaction mixture in a single continuous liquid phase; and

b) forming at least two substantially monoclonal nucleic acid populations by amplifying, by partially denaturing and without compartmentalizing and within the same reaction mixture of step (a) in the continuous liquid phase, each of the single polynucleotides within said at least two reaction sites, wherein the amplifying is performed using a second universal primer in solution that can hybridize to the second universal primer binding site within the polynucleotide templates, and wherein said at least two reaction sites are in fluid communication with each other during the amplifying.

2. The method of claim 1, wherein the at least two different polynucleotide templates have different sequences.

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

contacting the at least two polynucleotides with a single reaction mixture containing reagents for nucleic acid synthesis.

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

contacting the at least two polynucleotides with a recombinase in the different reaction sites.

5. The method of claim 1, wherein the at least two reaction sites are operatively coupled to a sensor.

6. The method of claim 5, wherein the sensor can detect the presence of a nucleotide incorporation byproduct within one or more of the reaction sites.

7. The method of claim 5, wherein one or more of the reaction sites includes a hydrophilic polymer matrix conformally disposed within the one or more reaction sites.

8. The method of claim 7, wherein the hydrophilic polymer matrix includes a hydrogel polymer matrix.

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

10. The method of claim 7, wherein the hydrophilic polymer matrix includes polyacrylamide, copolymers thereof, derivatives thereof, or combinations thereof.

11. The method of claim 10, wherein polyacrylamide is conjugated with an oligonucleotide primer.

12. The method of claim 7, wherein one or more of the reaction sites has a diameter in a range of 0.1 micrometers to 2 micrometers.

13. The method of claim 5, wherein the sensor includes a field effect transistor (FET).

14. The method of claim 13, further including detecting the presence of one or more nucleotide incorporation byproducts at one or more reaction sites of the array using the FET.

15. The method of claim 13, further including detecting a pH change occurring within the one or more reaction sites using the FET.

16. The method of claim 13, further including introducing a nucleotide into the one or more reaction sites;

and detecting an output signal from the sensor resulting from incorporation of the nucleotide into the sequencing primer.

17. The method of claim 16, wherein the output signal is based on a threshold voltage of the FET.

18. The method of claim 13, wherein the FET includes a floating gate conductor coupled to one or more of the reaction sites.

19. The method of claim 13, 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.

20. The method of claim 13, wherein the FET includes an ion sensitive FET (ISFET).