Nucleic Acid Sequence Analysis

• US 20100316999A1
• Filed: 01/04/2010
• Published: 12/16/2010
• Est. Priority Date: 07/28/1997
• Status: Abandoned Application

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Densham, Daniel Henry
• Application Number:
  US12/651,824
• Publication Number:
  US 20100316999A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2565/628 being a surface plasmon resonance spectrometer
• CPC Class Codes: C12Q 2565/633 NMR

Abstract

The present invention relates to a method for determining the sequence of a polynucleotide, the method comprising the steps of: (i) reacting a target polynucleotide with a polymerase enzyme immobilised on a solid support, and the different nucleotides, under conditions sufficient for the polymerase reaction; and (ii) detecting the incorporation of a specific nucleotide complementary to the target polynucleotide, by measuring radiation.

Claims

1. A method for sequencing a polynucleotide, comprising the steps of:

(i) reacting a target polynucleotide with a polymerase, wherein said polymerase is immobilized on a solid support, and nucleotides, under conditions sufficient for the polymerase reaction; and

(ii) detecting the interaction between the polymerase, the target polynucleotide, and each specific nucleotide complementary to the target polynucleotide that is incorporated into a nascent polynucleotide being synthesized as a result of the polymerase reaction, to thereby determine the sequence of the target polynucleotide, the detection being carried out by measuring a change in or absorption of radiation that occurs during the interaction.

2. The method according to claim 1, wherein steps (i) and (ii) are conducted with each of the complementary nucleotides in turn, until incorporation is detected, and then repeated.

3. The method according to claim 1, wherein step (i) is conducted with all the complementary nucleotides present.

4. The method according to claim 1, wherein the nucleotides comprise a 3′

blocking group which is removed after the polymerase reaction.

5. The method according to claim 4, wherein the blocking group is selectively removed by pulsed monochromatic light.

6. The method according to claim 4, wherein the nucleotides comprise a further blocking group at the terminal phosphate group of the triphosphate chain, and the further blocking group is removed prior to the removal of the 3′

blocking group.

7. The method according to claim 6, wherein the further blocking group is selectively removed by pulsed monochromatic light under conditions different from those required to remove the 3′

blocking group.

8. The method according to claim 7, wherein the further blocking group is removed by pulsing the monochromatic light for a duration different from that required to remove the 3′

blocking group.

9. The method according to claim 1, wherein step (i) further comprises introducing a competitive inhibitor of the polymerase.

10. The method according to claim 1, wherein the target polynucleotide of step (i) is bound to the polymerase enzyme by a β

₂ dimer complex.

11. The method according to claim 1, wherein the polymerase is E. coli DNA polymerase III or T7 polymerase.

12. The method according to claim 1, wherein the polymerase is a Taq polymerase.

13. The method according to claim 1, wherein the polymerase is reverse transcriptase.

14. The method according to claim 1, wherein step (ii) comprises detection of a change in resonance signal over time.

15. The method according to claim 1, wherein the radiation is electromagnetic.

16. The method according to claim 15, wherein the electromagnetic radiation is in the infra-red spectrum.

17. The method according to claim 1, wherein the incorporation of a nucleotide is detected using NMR.

18. The method according to claim 1, wherein the polynucleotide is DNA.

19. The method according to claim 1, wherein measurement is carried out using evanescent wave spectroscopy.

20. The method according to claim 1, wherein the nucleotides are not labeled.

21. The method according to claim 1, wherein the effect detected results from a conformation or mass change of the polymerase that occurs upon incorporation of the nucleotide.