EP0582256A2
Expired / Lapsed
EP
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Polynucleotide detecting method and apparatus
Assignee: Hitachi Ltd
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Filing: 1993-08-02
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Granted: —
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Expires: 2013-08-02
Abstract
Abstract Fluorescent-labeled probes 2 hybridizable with target polynucleotide chain 1 are used as fluorescent-labeled probes, and both are reacted to obtain a fluorescent-labeled probe 3 bound to the target polynucleotide 1. After electrophoretically separating the unreacted excess fluorescent-labeled probes, electrophoresis is further continued at a temperature above the denaturing temperature of the complex 3 of target polynucleotide and fluorescent-labeled probe to release the fluorescent-labeled probe 4 which has been bound to the target polynucleotide. Laser beam 6 is applied to the released fluorescent-labeled probe 4 and the generated fluorescence is detected by fluorescence detector 7. Thereby, the present invention enables successive automatic prosecution of a series of operating step and provides a high-sensitivity and high-precision quantitative polynucleotide detecting method suited for application to a high-throughput automated detecting apparatus.
Inventors
- 👤 Kazunori Okano
- 👤 Hideki Kambara
- 👤 Keiichi Nagai
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 23:38
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6823; G; G01; G01N; G01N27/00; G01N27/26; G01N27/416; G01N27/447; G01N27/44704; G01N27/44717; G01N27/44721
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
84 words
A polynucleotide detecting method comprising the steps of: (i) binding a fluorescent-labeled probe to a target polynucleotide under a hybridization condition to obtain a complex of said target polynucleotide and said fluorescent-labeled probe; (ii) electrophoretically separating away unreacted fluorescent-labeled probes from a mixture of said complex and unreacted fluorescent-labeled probes; (iii) denaturing said complex and electrophoretically separating the fluorescent-labeled probe which has been bound to said target polynucleotide; and (iv) exciting the fluorophore of said fluorescent-labeled probe to generate fluorescence and determining such fluorescence.
Claim 2
Dependent Claim
27 words
A method according to Claim 1, wherein a denaturing agent is added to the electrophoretic matrix used for the electrophoresis in step (iii) to denature said complex.
Claim 3
Dependent Claim
14 words
A method according to Claim 2, wherein the denaturing agent is urea or formamide.
Claim 4
Dependent Claim
34 words
A method according to Claim 1, wherein electrophoresis is carried out by denaturing said complex while raising, either continuously or stepwise, the temperature of the electrophoretic matrix used in the electrophoresis in step (iii).
Claim 5
Dependent Claim
44 words
A method according to Claim 1, wherein a denaturing agent is added to the electrophoretic matrix used in the electrophoresis in step (iii) and electrophoresis is carried out by denaturing said complex while raising, either continuously or stepwise, the temperature of said electrophoretic matrix.
Claim 6
Dependent Claim
32 words
A method according to Claim 1, wherein the electrophoretic matrix used for the electrophoresis in step (ii) and the electrophoretic matrix used for the electrophoresis in step (iii) are separate electrophoretic matrices.
Claim 7
Dependent Claim
67 words
A method according to Claim 1, wherein the electrophoretic matrix used for the electrophoresis in step (iii) is a gel or a membrane having pores not passing said target polynucleotide and made of a material selected from the group consisting of polyacrylamide, cellulose and fluorocarbon, and the electrophoretic matrix used for the electrophoresis in step (iii) is mainly composed of polyacrylamide, or no such matrix is used.
Claim 8
Dependent Claim
91 words
A polynucleotide detecting apparatus comprising: a first electrophoretic means for separating away unreacted fluorescent-labeled probes from a mixture of a complex of a target polynucleotide and fluorescent-labeled probe and the unreacted fluorescent-labeled probes, said complex having been obtained by binding the fluorescent-labeled probe to the target polynucleotide under a hybridization condition; a second electrophoretic means for denaturing said complex and separating the fluorescent-labeled probe bound to said target polynucleotide; a means for exciting the fluorophore of said fluorescent-labeled probe: and a means for detecting the fluorescence generated by said exciting means.
Claim 9
Dependent Claim
25 words
An apparatus according to Claim 8, wherein a denaturing agent is added to the electrophoretic matrix constituting said second electrophoretic means to denature said complex.
Claim 10
Dependent Claim
14 words
An apparatus according to Claim 9, wherein the denaturing agent is urea or formamide.
Claim 11
Dependent Claim
32 words
An apparatus according to Claim 8, wherein electrophoresis is carried out by denaturing said complex while raising, either continuously or stepwise, the temperature of the electrophoretic matrix constituting said second electrophoretic means.
Claim 12
Dependent Claim
42 words
An apparatus according to Claim 8, wherein a denaturing agent is added to the electrophoretic matrix constituting said second electrophoretic means and electrophoresis is carried out by denaturing said complex while raising, either continuously or stepwise, the temperature of said electrophoretic matrix.
Claim 13
Dependent Claim
28 words
An apparatus according to Claim 8, wherein the electrophoretic matrix constituting said first electrophoretic means and the electrophoretic matrix constituting said second electrophoretic means are separate electrophoretic matrices.
Claim 14
Dependent Claim
63 words
An apparatus according to Claim 8, wherein the electrophoretic matrix constituting said first electrophoretic means is a gel or a membrane having pores not passing said target polynucleotide and made of a material selected from the group consisting of polyacrylamide, cellulose and fluorocarbon, and the electrophoretic matrix constituting said second electrophoretic means is mainly composed of polyacrylamide, or no such matrix is used.
References Cited (Backward Citations)
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