Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-02-08
1998-10-20
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 231, 536 243, C12Q 168, C07H 2102, C07H 2104
Patent
active
058244743
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a method of detecting a nucleic acid employing the property of nucleic acids that polyamines bind thereto.
DESCRIPTION OF THE PRIOR ART
As a conventional method of detection of nucleic acids, methods using ethidium bromide have been widely adopted. The known method comprises intercalation of ethidium bromide into a nucleic acid so as to emit fluorescence by which a location of a nucleic acid is detected. However, the known method is very inconvenient because fluorescence cannot be detected in the light and the locations of nucleic acids can be detected only by corresponding original gel to a photograph thereof which were taken under ultraviolet radiation in the dark. And also, ethidium bromide requires careful handling because of its strong carcinogenic property (cf. Gene Operation Manual, page 2, lines 18-21, Kodansha Scientific Press). Alternatively, methods using anionic gold colloid and cationic iron colloid cacodylate are also known (Gene Anal. Techn. pages 1 to 5, 1986), however, the sensitivity of the methods is low. In addition, since the method using gold colloid is a kind of background staining system, detection of a nucleic acid is not easy and also hybridization after the staining is disadvantageously affected. In the method using iron colloid, there is a disadvantage that staining after hybridization cannot be carried out. The development of an operably easy, safe, simple and reproducible method for detecting a nucleic acid has been expected for a long time. It is already known that a polyamine can bind to DNA and also has affinity for RNA (cf. Biochemical Journal, 123, 811-815 (1967), Journal of Molecular Biology, 24, 113-122 (1967), ibid., 42, 363-373 (1969) and ibid., 121, 327-337 (1987)). And also, in order to detect target polynucleotides, it is known to use probes which bond covalently to polynucleotide complementary to protein modified with polyamine (Japanese Patent Publication (in Japanese) A: TOKUKOHYOSHO 60-501488 (1985), to which corresponds, Japanese Patent Publication B: TOKUKOHEI 2-59720 (1990) and its divisional application, Japanese Patent Publication A: TOKUKAIHEI 1-124400 (1989)). However, no method of detecting a nucleic acid is known which comprises forming a complex of a labeled polyamine and a nucleic acid.
SUMMARY OF THE INVENTION
The object of the present invention is thus to provide
(1) a method of detecting a nucleic acid which comprises bringing a solid carrier suspected to carry or contain a nucleic acid into contact with a polyamine to which a label capable of generating a detectable signal or a precursor thereof is bound, to form an electrostatically bonded complex between said nucleic acid and said polyamine, said precursor converting into said label, if used, removing the polyamine which has not formed any electrostatically bonded complex before or after the conversion of said precursor and then detecting said label, and
(2) a method of differentiating a target nucleic acid from any nucleic acid other than said target nucleic acid which comprises combining the following steps (i) and (ii) in voluntary order: contain a target nucleic acid, into contact with a probe to which a label capable of generating a detectable signal or a precursor thereof is bound, being capable of hybridizing with said target nucleic acid under hybridization conditions, to form a hybrid, said precursor converting into said label, if used, removing said probe which has not formed any hybrid before or after the conversion of said precursor and then detecting said label, and carry or contain a target nucleic acid, into contact with a polyamine to which a label capable of generating a detectable signal or a precursor thereof is bound, to form an electrostatically bonded complex between said nucleic acid and said polyamine, said precursor converting into said label, if used, removing said polyamine which has not formed any electrostatically bonded complex before or after the conversion of said precursor and then detecting said lab
REFERENCES:
patent: 4556653 (1985-12-01), Paau et al.
patent: 5053326 (1991-10-01), Renz
patent: 5599667 (1997-02-01), Arnold, Jr., et al.
Davis et al. Basic Methods in Molecular Biology. pp. 59-78 Elsvier Publishing, NY (1986).
The Stratagene Catalog p. 39 (1988 Edition).
Schmid et al. Location of Spermine and other polyamines on DNA as revealed by photoaffinity cleavage with polyaminobenziazonium salts. Biochemistry 30 :4357-4361 (1991).
Kishi Yuichiro
Matsuhisa Akio
Mikawa Yoshikazu
Shiba Kiyotaka
Fuso Pharmaceutical Industries Ltd.
Jones W. Gary
Whisenant Ethan
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