Method and apparatus for determining base sequence of nucleic acid molecule

• US 20060115833A1
• Filed: 09/22/2005
• Published: 06/01/2006
• 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
• Granted Patent: US 7,371,532 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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, said method comprising a first step of cleaving a nucleic acid molecule of interest while controlling the cleavage site, a second step of measuring the change in mass which occurs in the nucleic acid molecule after the first step, and a third step of acquiring the base information of the cleaved nucleic acid molecule from the data measured in the second step.

2. The method for determining the base sequence as defined in claim 1, wherein the first to third steps are repeated to determine the entire base sequence of the nucleic acid molecule of interest.

3. The method for determining the base sequence as defined in claim 1, wherein the first step employs at least a nuclease.

4. The method for determining the base sequence as defined in claim 2, wherein the nuclease is exonuclease.

5. The method for determining the base sequence as defined in claim 1, wherein the nucleic acid molecule of interest is a single-stranded nucleic acid molecule.

6. The method for determining the base sequence as defined in claim 1, wherein the first step is designed to cleave the nucleic acid molecule of interest by the unit of nucleic acid molecule.

7. The method for determining the base sequence as defined in claim 1, wherein the first step starts after the nucleic acid molecule of interest has been fragmented.

8. The method for determining the base sequence as defined in claim 7, wherein the base sequence of the fragmented nucleic acid molecules is determined in parallel at the same time.

9. The method for determining the base sequence as defined in claim 7, wherein the first and second steps are performed on oligonucleic acid molecules or polynucleic acid molecules composed only of identical base species so as to obtain information about the mass of one unit of nucleic acid molecule corresponding to each base species, and the thus obtained information is used to determine the base sequence of the nucleic acid molecule of interest.

10. The method for determining the base sequence as defined in claim 1, wherein the nucleic acid molecule of interest has its one end fixed to the surface of a solid phase.

11. The method for determining the base sequence as defined in claim 10, wherein the second step involves measuring the change in resonance frequency of a piezoelectric element.

12. The method for determining the base sequence as defined in claim 11, wherein the piezoelectric element is a quarts oscillator.

13. An apparatus for determining the base sequence of a nucleic acid molecule, said apparatus comprising a unit to supply a reaction region with a substance that cleaves a nucleic acid molecule of interest in said reaction region, a unit to detect the change in mass of the nucleic acid molecule which has undergone cleavage, a unit to analyze the data detected by said detecting unit, and a unit to send out the results of analysis accomplished by said analyzing unit.

14. The apparatus for determining the base sequence as defined in claim 13, wherein the analyzing unit is so constructed as to compare the detected data with the reference data which has previously been stored for individual base species.

15. The apparatus for determining the base sequence as defined in claim 13, wherein the detecting unit is so constructed as to detect the amplitude of vibration of the cantilever which permits the nucleic acid molecule of interest to be fixed to its end surface.