Highly sensitive nucleic acid hybridization method and gene analysis method by using the same

• US 20030186266A1
• Filed: 10/23/2002
• Published: 10/02/2003
• Est. Priority Date: 03/07/2001
• Status: Active Grant

• Current Assignee: NGK Insulators Limited
• Original Assignee: NGK Insulators Limited
• Inventors: Yamada, Kazunari, Miyazaki, Isao, Matsuda, Chiho, Kawase, Mituso
• Granted Patent: US 7,052,842 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2527/101 Temperature

Abstract

A method for hybridizing nucleic acids, which includes an annealing step of preparing a first single stranded nucleic acid fragment immobilized on a surface of an immobilizing material and a second single stranded nucleic acid fragment labeled with fluorescence or radioisotope and forming a complementary double strand from the first single stranded nucleic acid fragment and the second single stranded nucleic acid fragment, and an enzyme treatment step. In the annealing step, the complementary double strand is formed by performing a temperature gradient processing performed from a high temperature area to a low temperature area, and in the enzyme treatment step, a noncomplementary nucleic acid portion contained in the complementary double stand is recognized and cleaved by adding endonuclease. This method has high measurement sensitivity and the operation is simple.

Claims

1. A method for hybridizing nucleic acids, which comprises an annealing step of preparing a first single stranded nucleic acid fragment immobilized on a surface of an immobilizing material and a second single stranded nucleic acid fragment labeled with fluorescence or radioisotope and forming a complementary double strand from the first single stranded nucleic acid fragment and the second single stranded nucleic acid fragment, and an enzyme treatment step,

characterized in that

said complementary double strand is formed by a temperature gradient processing from a high temperature area to a low temperature area in said annealing step, and

an endonuclease is added to recognize and remove a noncomplementary nucleic acid portion contained in said complementary double stand in said enzyme treatment step.

2. The method for hybridizing nucleic acids according to claim 1, characterized in that, in said annealing step, a pH gradient processing is performed from a high pH area to a low pH area instead of the temperature gradient processing from a high temperature area to a low temperature area.

3. The method for hybridizing nucleic acids according to claim 1, characterized in that, in said annealing step, a salt concentration gradient processing is performed from a low salt concentration area to a high salt concentration area instead of the temperature gradient processing from a high temperature area to a low temperature area.

4. A method for gene analysis, characterized in that there is used the method for hybridizing nucleic acids according to any one of claims 1 to 3.