Heat and pH Measurement for Sequencing of DNA

• US 20080166727A1
• Filed: 12/18/2007
• Published: 07/10/2008
• Est. Priority Date: 12/20/2006
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Ronaghi, Mostafa, Esfandyarpour, Hesaam
• Granted Patent: US 7,932,034 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 of the DNA template;

b) placing multiple copies of the template DNA including the provided primer region in a reaction well, wherein the reaction well has a fluid volume of less than about 0.1 uL;

c) adding to the reaction well a DNA polymerization mixture containing DNA polymerase and a plurality of nucleotides; and

d) measuring a temperature change of at least 0.003°

C., whereby

incorporation of nucleotides by the DNA polymerase produces heat increase indicative of incorporation of a complementary nucleotide into the DNA template.

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

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

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

5. The method of claim 3 where 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 where the heat sensor is selected from the group consisting of a picocalorimeter a planar, one-dimensional thermocouple, a thermometer;

and an infra red (IR) sensor.

8. The method of claim 6 where the heat sensor is a planar sensor on a surface of the reaction well.

9. The method of claim 6 where the heat sensor is a thermocouple extending into the reaction well.

10. The method of claim 6 where 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 change of about 0.001 units, whereby a drop in pH indicates incorporation of a nucleotide complementary to the template.

12. The method of claim 1, further comprising adding to a plurality of reaction wells containing different template duplex combinations a DNA polymerization mixture containing DNA polymerase and at least one nucleotide.

13. The method of claim 11 wherein the plurality of reaction wells is contained in a single polymeric substrate provided with fluid channels.

14. The method of claim 1 where the primer region comprises a sample analyte.

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

16. A microfluidic device for determining DNA sequences, comprising:

a) a substrate having a plurality of reaction wells for holding DNA strands, DNA polymerase, and polymerization buffer;

b) fluid channels for delivering dNTPs to the reaction wells; and

c) a heat sensor connected individually to each reaction well for detecting temperature changes in each reaction well caused by incorporation of the dNTPs.

17. The device of claim 16 wherein the heat sensor comprises a picocalorimeter, a planar, one dimensional thermocouple, a thermometer or an infrared sensor.

18. The device of claim 18 further comprising an expandable control line adjacent to an opening in a reaction well for sealing the reaction well from the fluid channel by expansion of the control line to a size greater than the opening.

19. A microfluidic device for determining DNA sequences, comprising:

a) a substrate having a plurality of reaction wells for holding DNA strands, DNA polymerase, and polymerization buffer;

b) fluid channels for delivering dNTPs to the reaction wells; and

c) a pH sensor connected individually to each reaction well for detecting pH changes in each reaction well caused by incorporation of the dNTPs.

20. The device of claim 19 wherein the pH sensor is selected from the group consisting of a micromechanical cantilever, and an electronic pH meter.

21. The device of claim 19 further comprising a control line adjacent to a reaction well for sealing the reaction well from the fluid channel.