Droplet-Based Nucleic Acid Amplification in a Temperature Gradient

• US 20100323405A1
• Filed: 06/23/2008
• Published: 12/23/2010
• Est. Priority Date: 06/22/2007
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Paik, Philip, Eckhardt, Allen, Pollack, Michael G.
• Granted Patent: US 8,951,732 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/15 Gradients

Abstract

A method of amplifying a nucleic acid, the method comprising cycling an amplification-ready droplet through a temperature gradient to locations within the temperature gradient suitable for effecting steps in an amplification reaction.

Claims

1. A method of amplifying a nucleic acid, the method comprising cycling an amplification-ready droplet through a temperature gradient to locations within the temperature gradient suitable for effecting steps in an amplification reaction.

2. The method of claim 1 wherein the droplet is shuttled through an oil-based filler fluid on a droplet actuator to the locations.

3. The method of claim 1 wherein the locations comprise locations having temperatures suitable for:

(a) adjusting the droplet to a temperature selected to denature double stranded nucleic acid in the droplet into single-stranded DNA (typically about 90-1000 C);

(b) adjusting the droplet temperature selected to permit primers to anneal to their complementary sequences on the nucleic acid template strands (typically about 50-750 C); and

(c) adjusting the droplet temperature selected to facilitate extension of the double stranded segment of the nucleic acid to be extended by incorporation of additional nucleotides (typically about 70-750 C).

4. The method of claim 1 wherein the method comprises shuttling the droplet to different locations within the temperature gradient and identifying improved amplification patterns based on the results of varying the locations.

5. The method of claim 4 comprising independently varying anneal location to identify an improved anneal location or temperature.

6. The method of claim 4 comprising independently varying extension location to identify an improved extension location or temperature.

7. The method of claim 4 comprising independently varying denaturation location to identify an improved denaturation location or temperature.

8. The method of claim 4 further comprising executing an improved PCR amplification protocol using the newly identified locations.

9. The method of claim 4 further comprising minimizing denaturation and annealing/extension times to achieve minimum analysis time.

10. The method of claim 1 wherein the temperature gradient is established using a heating element arranged to heat filler fluid at a locus of an electrode path.

11. The method of claim 1 wherein the temperature gradient is established comprising:

(a) using a heating element arranged to heat filler fluid at a first locus of an electrode array or path; and

(b) a heat sink element arranged to heat filler fluid at a second locus of an electrode array or path.

12. The method of claim 1 further comprising mapping the temperature gradient of the droplet actuator.

13. The method of claim 1 further comprising mapping the temperature gradient of the droplet actuator to specific electrodes on a path or array of electrodes on the droplet actuator.