Method and apparatus for determining base sequence of nucleic acid molecule

• US 7,371,532 B2
• Filed: 09/22/2005
• Issued: 05/13/2008
• Est. Priority Date: 09/28/2004
• Status: Active Grant

• Current Assignee: Sony Corporation (Sony Group Corp.)
• Original Assignee: Sony Corporation (Sony Group Corp.)
• Inventors: Yamamoto, Takuro, Mamine, Takayoshi, Tsujimoto, Atsumi
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/231,998
• Publication Number:
  US 20060115833A1
• Time in Patent Office: 964 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

Disclosed herein is a method and apparatus for determining the base sequence of a nucleic acid molecule by cleaving a nucleic acid molecule of interest while controlling the cleavage site, measuring the change in mass which occurs in the nucleic acid molecule after the cleavage step, and acquiring the base information of the cleaved nucleic acid molecule from the data about the change in mass. The method and apparatus are based on the principle which is entirely different from that used for the conventional technique.

Claims

1. A method for determining the base sequence of a nucleic acid molecule, wherein the nucleic acid molecule is immobilized on a cantilever comprising a piezoelectric element, said method comprising

a) measuring the frequency of vibration of the piezoelectric element;

b) cleaving the nucleic acid molecule;

c) measuring the frequency of vibration of the piezoelectric element; and

d) determining the sequence of the nucleic acid molecule by comparing the frequency measured in a) with the frequency measured in c).

2. The method of claim 1, wherein are repeated to determine the entire base sequence of the nucleic acid molecule.

3. The of claim 1, wherein a) employs at least a nuclease.

4. The method of claim 3, wherein the nuclease is an exonuclease.

5. The method of claim 1, wherein the nucleic acid molecule is single-stranded.

6. The method for determining the base sequence of claim 1, wherein b) comprises cleaving a single nucleotide from the nucleic acid molecule.

7. The method of claim 1, wherein the nucleic acid molecule of b) comprises a portion of a larger nucleic acid molecule that has been fragmented.

8. The method for determining the base sequence of claim 7, wherein the base sequences of multiple portions of a larger nucleic acid molecule that has been fragmented are determined in parallel.

9. The method of claim 1, wherein a-c are performed on oligonucleic or polynucleic acid molecules comprising identical bases to determine the mass of each base.

10. The method of claim 1, wherein the piezoelectric element is a quartz oscillater.

11. The method of claim 1, wherein the nucleic acid molecule is biotinylated and immobilized on a cantilever comprising streptavidin.

12. The method of claim 3, wherein the nuclease is selected from deoxyribonuclease, ribonuclease, NNase-I, NNase-II, ATP-dependent NNase, RNase-III, nuclease S₁, nuclease P₁, exoNNase I, RNase II, RNase T₁, RNase U₂, RNase A, T4-endonuclease, RNase H, RNase P, and restriction enzymes.

13. The method of claim 1, wherein the cantilever comprises a ferroelectric substance.

14. The method of claim 13, wherein the ferroelectric substance is selected from lead zirconate titanate (PZT) and strontium bismuth tantalate (SBT).