Heat and pH measurement for sequencing of DNA

• US 7,932,034 B2
• Filed: 12/18/2007
• Issued: 04/26/2011
• Est. Priority Date: 12/20/2006
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Ronaghi, Mostafa, Esfandyarpour, Hesaam
• Primary Examiner(s): Kim; Young J
• Assistant Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US11/959,317
• Publication Number:
  US 20080166727A1
• Time in Patent Office: 1,225 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

The present method involves sequencing by synthesis in which a template strand having an attached primer is immobilized in a small volume reaction mixture. In one embodiment, the reaction mixture is in contact with a sensitive heat sensor, which detects the heat of reaction from incorporation of a complementary base (dNTP) in the presence of appropriate reagents (DNA polymerase, and polymerase reaction buffer). Alternatively, or in addition, a change in pH resulting from the incorporation of nucleotides in the DNA polymerase reaction is measured. A device is provided having delivery channels for appropriate reagents, including dNTPs, which may be delivered sequentially or in a mixture. Preferably, the dNTPs are added in a predetermined sequence, and the dNTP is incorporated or not depending on the template sequence.

Claims

1. A method for obtaining sequence information from a single-stranded DNA template comprising:

a) providing a primer region hybridized to the single-stranded DNA template, wherein the primer region is;

(i) a hairpin formed by self-hybridization of the single-stranded DNA template, or (ii) a single-stranded oligonucleotide that is complementary to a region of the single-stranded DNA template;

b) placing multiple copies of the single-stranded DNA template including the provided primer region in reaction chambers in a microfluidic device comprising a plurality of reaction chambers that are thermally isolated from each other, wherein the reaction chambers have a fluid volume of less than 0.1 μ

L of reaction mixture;

c) adding to said reaction chambers containing said copies a mixture containing DNA polymerase and a plurality of a single species of nucleotides;

d) removing unincorporated nucleotides;

e) measuring a temperature change in said reaction chambers containing said copies of at least 0.003°

C., said temperature change being measured by a probe in proximity to the reaction mixture as a result of the incorporation of nucleotides by the DNA polymerase producing a heat increase indicative of the incorporation of a complementary nucleotide into the primer region hybridized to the single-stranded DNA template; and

f) repeating steps c), d), and e) to obtain the sequence information.

2. The method of claim 1, further comprising immobilizing the single-stranded DNA template.

3. The method of claim 2, wherein the immobilizing comprises immobilizing the single-stranded DNA template on a bead, which has a diameter at least half of the diameter of an opening in the reaction chamber.

4. The method of claim 3, wherein the bead is made of metal.

5. The method of claim 3 wherein the bead is a magnetic bead approximately 2-3 μ

m in diameter.

6. The method of claim 1, wherein the measuring of the temperature change is accomplished using a heat sensor.

7. The method of claim 6 wherein the heat sensor is selected from the group consisting of a picocalorimeter, a planar one-dimensional thermocouple, a thermometer, and an infrared (IR) sensor.

8. The method of claim 6 wherein the heat sensor is a planar sensor on a bottom surface of the reaction chamber.

9. The method of claim 6 wherein the heat sensor is a thermocouple extending into a bottom portion of the reaction chamber.

10. The method of claim 6 wherein the heat sensor is an IR detector in an IR microscope.

11. The method of claim 1 further comprising the step of measuring a change in pH of about 0.001 units, said change in pH comprising a drop in pH that indicates the incorporation of a nucleotide complementary to the single-stranded DNA template into the primer.

12. The method of claim 1, wherein different reaction chambers in the plurality of thermally isolated reaction chambers contained in the microfluidic device contain primer regions hybridized to single-stranded DNA templates that differ in sequence between reaction chambers.

13. The method of claim 12 wherein the plurality of reaction chambers is contained in a single polymeric substrate provided with fluid channels.

14. The method of claim 1 wherein the primer region comprises a single-stranded oligonucleotide that is complementary to a region of the single-stranded DNA template.

15. The method of claim 1 wherein the reaction mixture is between about 70 picoliters and about 30 femtoliters in volume.

16. The method of claim 1 wherein said multiple copies of the single-stranded DNA template in a reaction chamber comprise at least 10³ copies.

17. The method of claim 1 wherein the sequence information comprises 80 to 120 bases.

18. The method of claim 11 further comprising the step of splitting pyrophosphate (PPi) to 2Pi.