NUCLEIC ACID AMPLIFICATION

• US 20140080717A1
• Filed: 09/10/2013
• Published: 03/20/2014
• 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, HINZ, Wolfgang
• Granted Patent: US 9,334,531 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 polynucleotides into an array of reaction chambers by introducing a single one of said polynucleotides into at least two of said reaction chambers; and

(b) forming at least two substantially monoclonal nucleic acid populations by amplifying the polynucleotides within said at least two reaction chambers, wherein the said at least two reaction chambers are in fluid communication with each other during the amplifying.

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

3. The method of claim 1, wherein the amplifying is performed without completely denaturing the at least two polynucleotides during the amplifying.

4. 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.

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

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

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

7. The method of claim 6, wherein the sensor can detect the presence of a nucleotide incorporation byproduct within the reaction chamber.

8. The method of claim 6, wherein the reaction chamber includes a hydrophilic polymer matrix conformally disposed within the reaction chamber.

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

10. The method of claim 8, wherein the hydrophilic polymer matrix is a cured-in-place polymer matrix.

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

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

13. The method of claim 8, wherein the reaction chamber has a characteristic diameter in a range of 0.1 micrometers to 2 micrometers.

14. The method of claim 6, wherein the sensor includes a field effect transistor (FET). The FET can include an ion sensitive FET (ISFET).

15. The method of claim 14, further including detecting the presence of one or more nucleotide incorporation byproducts at a site of the array using the FET.

16. The method of claim 14, further including detecting a pH change occurring within the at least one reaction chamber using the FET.

17. The method of claim 14, further including introducing a nucleotide into the site;

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

19. The method of claim 14, wherein the FET includes a floating gate conductor coupled to the reaction chamber.

20. The method of claim 14, 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 18, wherein the output signal is optionally based on a threshold voltage of the FET.