Apparatus for detecting denaturation of nucleic acid

• US 5,599,504 A
• Filed: 07/15/1994
• Issued: 02/04/1997
• Est. Priority Date: 07/15/1993
• Status: Expired due to Fees

• Current Assignee: Hamamatsu Photonics KK
• Original Assignee: Hamamatsu Photonics KK
• Inventors: Fukami, Tadashi, Tsuji, Akihiko, Hosoi, Shigeru
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): Ludlow, Jan M.
• Application Number:
  US08/275,910
• Time in Patent Office: 935 Days
• Field of Search: 422/82.08, 422/68.1, 422/82.05, 436/55, 436/94, 435/6, 435/91.1, 435/91.2, 435/287.2, 435/288.7, 935/85-88
• US Class Current: 422/82.08
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

An apparatus for detecting denaturation of a nucleic acid, including: denaturation condition controlling element for controlling condition of denaturation under which a double-strand nucleic acid is separated into a first single-strand nucleic acid and a second single-strand nucleic acid; excitation light irradiation source for irradiating the double-strand nucleic acid before denaturation, and the first single-strand nucleic acid and second single-strand nucleic acid after the denaturation; fluorescence detection monitor for detecting fluorescence emission based on the excitation light irradiation; and processing element for receiving, storing and processing a signal supplied from the fluorescence detection monitor.

Claims

1. An apparatus for detecting denaturation of a nucleic acid, comprising:

first nucleic acid amplifying means for separating a predetermined site of a first double-stranded nucleic acid to be examined into a first single-stranded nucleic acid and a second single-stranded nucleic acid and producing a plurality of a third single-stranded nucleic acid and a fourth single-stranded nucleic acid, said third single-stranded nucleic acid comprising a base or nucleotide provided with a first label molecule and having a base sequence complementary to that of the first single-stranded nucleic acid, said fourth single-stranded nucleic acid having a base sequence complementary to that of the second single-stranded nucleic acid;

second nucleic acid amplifying means for separating a predetermined site of a second double-stranded nucleic acid as a reference, which is the same or substantially the same as the first double-strand nucleic acid, into a fifth single-stranded nucleic acid which is the same or substantially the same as the first single-stranded nucleic acid, and a sixth single-stranded nucleic acid which is the same as or substantially the same as the second single-stranded nucleic acid, and produceing a plurality of a seventh single-stranded nucleic acid and an eighth single-stranded nucleic acid;

said seventh single-stranded nucleic acid comprising a base or nucleotide provided with a second label molecule which is capable of causing energy transfer between itself and the first label molecule located within a predetermined distance, and having a base sequence complementary to that of the fifth single-stranded nucleic acid;

said eighth single-stranded nucleic acid having a base sequence complementary to that of the sixth single-stranded nucleic acid;

nucleic acid mixing means for mixing the third single-stranded nucleic acid and the eighth single-stranded nucleic acid after the extraction thereof to produce a third double stranded or partially double-stranded nucleic acid;

denaturation condition controlling means for controlling a condition for denaturation under which the third double-stranded nucleic acid is separated into the third single-stranded nucleic acid and the eighth single-stranded nucleic acid;

irradiation means for emitting excitation light having a predetermined wavelength to irradiate the third double-stranded nucleic acid before denaturation by the denaturation controlling means, and the third single-stranded nucleic acid and eighth single-stranded nucleic acid after denaturation by the denaturation controlling means;

fluorescence detection means for detecting fluorescence emission based on the excitation light irradiation which emission is capable of being changed depending on a change in the amount of energy transfer between the first label molecule and the second label molecule, whereby a denaturation point is determined on the basis of the change in the fluorescence emission in response to the change in the denaturation condition; and

processing means for receiving, storing and processing a signal supplied from the fluorescence detection means;

whereby the fluorescence emitted from the nucleic acid is observed while changing the denaturation condition by the denaturation condition controlling means to measure the denaturation condition of the third double-stranded nucleic acid.

2. An apparatus for detecting denaturation of a nucleic acid according to claim 1, wherein the nucleic acid mixing means mixes the third single-stranded nucleic acid and the eighth single-stranded nucleic acid nucleic acid after the extraction thereof.

3. An apparatus for detecting denaturation of a nucleic acid according to claim 1, wherein the denaturation condition is temperature and the denaturation condition controlling means comprises a temperature controller.

4. An apparatus for detecting denaturation of a nucleic acid according to claim 1, wherein the denaturation condition is hydrogen ion concentration and the denaturation condition controlling means comprises a hydrogen ion concentration controller.

5. An apparatus for detecting denaturation of a nucleic acid according to claim 1, wherein the denaturation condition is the concentration of a denaturing agent capable of denaturing the third double-strand nucleic acid and the denaturation condition controlling means comprises a denaturing agent concentration controller.

6. An apparatus for detecting denaturation of a nucleic acid according to claim 1, wherein the fluorescence detection means selectively detects a first fluorescence signal which is emitted from the first label molecule and is capable of being changed depending on a change in the amount of energy transfer between the first label molecule and the second label molecule, and the processing means calculates a denaturation point on the basis of the change in the first fluorescence signal in response to the change in the denaturation condition.

7. An apparatus for detecting denaturation of a nucleic acid according to claim 1, wherein the fluorescence detection means selectively detects a second fluorescence signal which is emitted from the second label molecule and is capable of being changed depending on a change in the amount of energy transfer between the first label molecule and the second label molecule, and the processing means calculates a denaturation point on the basis of the change in the second fluorescence signal in response to the change in the denaturation condition.

8. An apparatus for detecting denaturation of a nucleic acid according to claim 1, wherein the fluorescence detection means detects the lifetime of fluorescence which is capable of being changed depending on a change in the amount of energy transfer between the first label molecule and the second label molecule, and the processing means calculates a denaturation point on the basis of the change in the lifetime of the fluorescence in response to the change in the denaturation condition.

9. An apparatus for detecting denaturation of a nucleic acid according to claim. 1, wherein the fluorescence detection means detects the spectrum of fluorescence which is capable of being changed depending on a change in the amount of energy transfer between the first label molecule and the second label molecule, and the processing means calculates a denaturation point on the basis of the change in the spectrum of the fluorescence in response to the change in the denaturation condition.

10. An apparatus for detecting denaturation of a nucleic acid according to claim 9, wherein the fluorescence detection means comprises a fluorescence spectrum measuring device.

11. An apparatus for detecting denaturation of a nucleic acid according to claim 10, wherein the fluorescence detection means comprises a fluorescence spectrum measuring device of a time-resolved type.