Method of discriminating a kind of nucleic acids bases

• US 5,322,796 A
• Filed: 11/02/1992
• Issued: 06/21/1994
• Est. Priority Date: 10/31/1991
• Status: Expired due to Fees

• Current Assignee: Hamamatsu Photonics KK
• Original Assignee: Hamamatsu Photonics KK
• Inventors: Ishikawa, Mitsuru
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): BHAT, NINA NMN
• Application Number:
  US07/968,868
• Time in Patent Office: 596 Days
• Field of Search: 436/96, 436/94, 536/27, 435/6, 935/77
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/145555 Hetero-N

Abstract

A method for discriminating four kinds of nucleic acid bases of DNA at high speed by utilizing chromophores intrinsic to DNA. The sample is added to a polar glassy solvent. Then the temperature of the glassy solution is reduced. A (n,π*) quencher is then added as a fluorescence intensifying reagent to the solution. The solution is then irradiated with an excitation light of UV laser beams thereto. The lifetimes of fluorescence from the sample are measured and used to discriminate the nucleic acid bases of DNA.

Claims

1. A method for discriminating four kinds of nucleic acid bases of DNA, adenine, thymine, guanine and cytosine, comprising the steps of:

adding a sample to a polar vitreous solvent to form a glassy solution, said polar vitreous solvent comprising a polar solvent selected from the group consisting of alcohol, a mixed liquid of alcohol and water in an arbitrary mixing ratio, and a mixed liquid of alcohol and ether or ketone in an arbitrary mixing ratio;

reducing the temperature of the glassy solution;

adding an (n,π

*) quencher comprising a strong acid as a fluorescence intensifying agent to the solution;

irradiating an excitation light of UV laser beams thereto;

measuring the lifetime of fluorescence from the sample; and

discriminating the kind of the nucleic acid base of DNA included in the sample, based on the lifetime of the fluorescence from the sample.

2. A method according to claim 1, wherein said an (n,π

*) quencher is a strong acid having no absorption spectrum in the wavelength region of the excitation light of 250-290 nm.

3. A method according to claim 2, wherein said strong acid is 0.1N hydrochloric acid.

4. A method according to claim 1, wherein the kind of the nucleic acid base is discriminated based on average fluorescence lifetime of the sample.