DNA ANALYSIS APPARATUS

• US 20090253194A1
• Filed: 04/07/2009
• Published: 10/08/2009
• Est. Priority Date: 04/08/2008
• Status: Active Grant

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: KAMBARA, Hideki, KISHIMOTO, Akihiko
• Granted Patent: US 8,975,066 B2
• Time in Patent Office: Days
• US Class Current: 435/287.200
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

Accurate and sensitive sequencing in pyrosequencing is achieved by allowing complementary strand synthesis reaction to proceed homogeneously and completely in a short time while performing luminescence reaction for a sufficiently long time. DNA as a sequencing target is immobilized on the surface of a solid supporter. Nucleic acid substrates are injected from a dispenser to the supporter site where complementary strand synthesis is in turn performed rapidly and completely in a short time under a small reaction volume. Next, the supporter together with the product thereon is moved into a luminescence reaction solution where luminescence reaction is in turn performed. Thus, a DNA complementary strand synthesis reaction site and a luminescence reaction site are completely separated. The-supporter surface is also washed by dipping the supporter in the luminescence reaction solution that contains a luminescence reagent and an enzyme that degrades redundant nucleic acid substrates.

Claims

1. A DNA analysis apparatus, comprising:

a holding member which holds a DNA sample;

a substrate supplying part which individually supplies plural types of substrates for complementary strand synthesis to the DNA sample held by the holding member;

a reaction vessel which accommodates a reaction solution that produces luminescence through reaction with a DNA complementary strand synthesis reaction product;

a photodetector which detects luminescence produced in the reaction solution; and

a controlling part which controls the position of the holding member as well as the supply of the substrates for complementary strand synthesis from the substrate supplying part, wherein

the controlling part positions the holding member outside the reaction solution in the reaction vessel to perform the supply of the substrates for complementary strand synthesis from the substrate supplying part and then moves the holding member into the reaction solution in the reaction vessel.

2. The DNA analysis apparatus according to claim 1, wherein the reaction solution is prepared in large excess relative to the substrates for complementary strand synthesis supplied from the substrate supplying part.

3. The DNA analysis apparatus according to claim 1, wherein the holding member is a membrane.

4. The DNA analysis apparatus according to claim 1, wherein the holding member is a string-like member, beads, or magnetic beads.

5. The DNA analysis apparatus according to claim 1, wherein the DNA complementary strand synthesis reaction product is pyrophosphoric acid.

6. The DNA analysis apparatus according to claim 1, wherein the controlling part controls the position of the holding member through the on/off control of magnetic force.

7. The DNA analysis apparatus according to claim 1, wherein the DNA sample is held by the holding member either through direct immobilization or via an enzyme for complementary strand synthesis immobilized on the holding member.

8. The DNA analysis apparatus according to claim 1, wherein after the supply of the substrates for complementary strand synthesis from the substrate supplying part, a reagent for degrading the substrates for complementary strand synthesis is supplied to the DNA sample.

9. The DNA analysis apparatus according to claim 1, wherein in the holding member, a region which holds an enzyme or catalyst that degrades the substrates for complementary strand synthesis is disposed adjacent to a region which holds the DNA sample.

10. The DNA analysis apparatus according to claim 1, wherein the holding member is a membrane, wherein a membrane which holds the DNA sample and a membrane which holds an enzyme or catalyst that degrades the substrates for complementary strand synthesis are superposed.

11. The DNA analysis apparatus according to claim 1, wherein the holding member is a membrane, wherein a region which holds the DNA sample and a region which holds an enzyme or catalyst that degrades the substrates for complementary strand synthesis are disposed in the same plane of the membrane.

12. The DNA analysis apparatus according to claim 11, wherein the region which holds the DNA sample is disposed in a central part of the membrane, and the region which holds an enzyme or catalyst that degrades the substrates for complementary strand synthesis is disposed in a peripheral part thereof.

13. The DNA analysis apparatus according to claim 1, wherein in the holding member, a region which holds the DNA sample is disposed upstream from a site to which the substrates for complementary strand synthesis are supplied, and a region which holds an enzyme or catalyst that degrades the substrates for complementary strand synthesis is disposed downstream from the site.

14. The DNA analysis apparatus according to claim 13, wherein the apparatus further comprises a section which allows a washing solution to run from the region which holds the DNA sample toward the region which holds an enzyme or catalyst that degrades the substrates for complementary strand synthesis, after the supply of the substrates for complementary strand synthesis from the substrate supplying part.

15. The DNA analysis apparatus according to claim 1, wherein the substrate supplying part supplies the substrates for complementary strand synthesis to the DNA sample from a reagent reservoir by use of a capillary or microfabrication device.

16. The DNA analysis apparatus according to claim 1, wherein the substrate supplying part and the reaction vessel are relatively fixed, and the holding member is moved relatively to the substrate supplying part and the reaction vessel.

17. The DNA analysis apparatus according to claim 1, wherein the substrate supplying part and the holding member are relatively fixed, and the substrate supplying part and the holding member are moved relatively to the reaction vessel.