DNA analysis method and DNA analyzer

• US 8,246,810 B2
• Filed: 04/08/2011
• Issued: 08/21/2012
• 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): Olsen, Kaj K
• Assistant Examiner(s): CARLSON, KOURTNEY SALZMAN
• Application Number:
  US13/064,682
• Publication Number:
  US 20110278179A1
• Time in Patent Office: 501 Days
• Field of Search: 20440301-40315, 205/777.5, 205/778, 205/792, 435/6, 435/91.1, 435/287.2, 435/6.11
• US Class Current: 205/777.5
• 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 deoxyribonucleic acid (DNA) analysis method comprising the steps of:

providing a sample containing a DNA to be analyzed and a primer hybridized to the DNA;

carrying out an extension reaction of the primer in the presence of deoxyribonucleotide triphosphates (dNTPs) and a DNA polymerase so that a primer extension reaction product is formed;

converting pyrophosphoric acid formed as a result of the extension reaction into a redox compound; and

electrically detecting the redox compound by detecting a variation in interference potential of an electrode in contact with the sample.

2. The DNA analysis method of claim 1, wherein a variation in interface potential of the electrode is detected using a field-effect transistor, wherein the field-effect transistor comprises a gate terminal electrically connected to the electrode, and wherein the field-effect transistor and the electrode are arranged on or above the same substrate.

3. The DNA analysis method of claim 1, wherein the dNTPs are deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP), or sulfur-containing derivatives of the dNTPs.

4. The DNA analysis method of claim 3, wherein one of the dNTPs is a sulfur-containing deoxyribonucleotide triphosphate (dNTP).

5. The DNA analysis method of claim 1, wherein an electrochemically active material is immobilized to the electrode through an insulating molecule.

6. The DNA analysis method of claim 5, wherein the insulating molecule comprises a carbon chain.

7. The DNA analysis method of claim 5, wherein the redox compound is oxidized nicotinamide adenine dinucleotide (NAD), and wherein the electrochemically active material is a quinone-containing molecule.

8. The DNA analysis method of claim 5, wherein the redox compound is a metal complex, and wherein the electrochemically active material is a ferrocene-containing material.

9. The DNA analysis method of claim 5, further comprising the step of carrying out washing a vessel for measuring with a washing buffer before carrying out said extension reaction.

10. The DNA analysis method of claim 9, wherein the washing buffer comprises a redox compound for initialization.

11. The DNA analysis method of claim 10, wherein the washing buffer comprises thiol compound, hydrogen peroxide, or potassium ferrocyanide.