DNA analysis method and DNA analyzer

• US 7,960,113 B2
• Filed: 02/04/2008
• Issued: 06/14/2011
• Est. Priority Date: 03/23/2007
• Status: Expired due to Fees

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Ishige, Yu, Kamahori, Masao
• Primary Examiner(s): Nguyen; Nam X
• Assistant Examiner(s): CARLSON, KOURTNEY SALZMAN
• Application Number:
  US12/068,206
• Publication Number:
  US 20090166221A1
• Time in Patent Office: 1,226 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

Disclosed is a DNA analysis method and a DNA analyzer whose signal intensity is not lowered even when a material at a higher density is measured. There is supplied dATP, dTTP, dGTP, or dCTP from a dATP solution vessel, a dTTP solution vessel, a dGTP solution vessel, or a dCTP solution vessel, and this causes an extension reaction of a double-stranded DNA immobilized to a bead, to yield pyrophosphoric acid. The pyrophosphoric acid is converted into a redox compound by the actions of a reagent and an enzyme contained in a reaction buffer in a reaction buffer vessel. The redox compound causes a variation in surface potential of a measuring electrode bearing an electrochemically active material immobilized thereto through an insulating molecule, and this variation causes a variation in drain current of a field-effect transistor electrically connected to the measuring electrode. Thus, the extension reaction is detected.

Claims

1. A DNA analysis system comprising:

a vessel bearing an immobilized nucleic acid or containing an article bearing an immobilized nucleic acid, wherein the vessel is configured to receive a liquid;

a reagent in the vessel, which converts pyrophosphoric acid formed as a result of an extension reaction of the nucleic acid into a redox compound;

one or more measuring electrodes that are in contact with the liquid to be contained in the vessel for detecting the redox compound;

an electrochemically active material that is immobilized to a surface of the measuring electrode through an insulating molecule;

a reference electrode that is in contact with the liquid;

one or more potentiometers that measure interface potentials of the one or more measuring electrodes;

a first feeder that feeds dATP or an analogue thereof to the vessel;

a second feeder that feeds dGTP or an analogue thereof to the vessel;

a third feeder that feeds dCTP or an analogue thereof to the vessel;

and a fourth feeder that feeds dTTP or an analogue thereof to the vessel.

2. The DNA analysis system of claim 1,

wherein the article bearing the immobilized nucleic acid is a bead having a radius r and a specific surface area R,

wherein molecules of the nucleic acid are immobilized on the bead at intervals d,

wherein the one or more potentiometers each have a lower measuring limit in concentration of a redox compound C_lim, and the Avogadro'"'"'s number is represented by N_A, and

wherein the following condition is satisfied:

r≦

3RC_lim/{(6−

π

)d²N_A}.

3. The DNA analysis system of claim 1, wherein the one or more potentiometers comprise one or more field-effect transistors.

4. The DNA analysis system of claim 3,

wherein the measuring electrodes and the field-effect transistors are arranged on or above on the same substrate.

5. The DNA analysis system of claim 1, wherein the electrochemically active material comprises a quinone-containing molecule.

6. The DNA analysis system of claim 1, wherein the electrochemically active material comprises a ferrocene-containing material.

7. The DNA analysis system of claim 1, further comprising a cleaning unit that feeds a washing buffer into the vessel.

8. The DNA analysis system of claim 7, wherein the washing buffer comprises a redox compound for initialization.

9. The DNA analysis system of claim 1, further comprising a unit that feeds a reaction buffer into the vessel, wherein the reaction buffer comprises the reagent which converts pyrophosphoric acid formed as a result of an extension reaction of the nucleic acid into a redox compound.