1987 Fiscal Year Final Research Report Summary
INNOVATION OF A NON-RADIO-ACTIVE LABELLING SUBSTANCE FOR NUVLEIC ACIDS.
| Project/Area Number |
60840019
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
遺伝学
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
HORR Hiroshi Fac.ofSci. HOKKAIDO UNIVERSITY, PROFESSOR, 理学部, 教授 (40000814)
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| Co-Investigator(Kenkyū-buntansha) |
KIMURA Masahito Fac.of Sci HOKKAIDO UNIVERSITY, ASSOCIATE, 理学部, 助手 (30091440)
TAKAGI Nobuo HOKKAIDO UNIVERSITY, ASSISTANT PROFESSOR, 遺伝子実験施設, 助教授 (20001852)
IWABUCHI Masaki Fac. of Sci. HOKKAIDO UNIVERSITY, ASSISTANT PROFESSOR, 理学部, 助教授 (30000839)
MIURA Kazunobu Fac.of phar Sci HOKKAIDO UNIVERSITY, ASSOCIATE, 薬学部, 助手 (70001980)
OHTSUKA Eiko Fac.ofPhar.sci HOKKAIDO UNIVERSITY, PROFESSOR, 薬学部, 教授 (80028836)
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| Project Period (FY) |
1985 – 1987
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| Keywords | NUCLEIC ACID DETECTION / FLUOROCHROME / 遺伝子 |
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| Research Abstract |
In recombinant DNA techniques, it is requires to detect a vety small quantity of nucleic acids, and in standard protocols this is accomplished by the use of rasiolabeled nucleic acids as probes. However, the inherent drawbacks of radiolabeled probes, such as chemical lability due to radiolytic decomposition, personnel safety and disposal problems, short haif-life and duration of autoradiographic exposure limit the usefulness of radio-labeled probes in conventional laboratories. It is therefore desirable to have sensitive, non-radioisotope methods for detecting nucleic acids.
Recently, efforts have been made in a number of laboratories to innovate a new non-radio-isotopic methods and a variety of methods have been proposed. Among these, the AB method which utilizes the specific and strong affinity of streptavidin and biotin appears to be the most sensitive and reliable one. With thie technique, it is possible to detect a unique sequence present in l ug of genomic DNA. However, the sensit
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ivity is still lower than that with radiolabeled probes. To improve the sensitivity of the AB technique, we attempted to synthesize fluorogenic substrates for alkaline phosphatase which upon hydrolysis yield insoluble, highly fluorogenic products.
One derivative of dibenzofuran and twenty derivatives of coumarine have been synthesized and tested for usefulness. Biotinylated lambda phage DNA was spotted on strips of nylon membrane, hybridized with strepavidin-alkaline phoshatase complex and incubated in a medium containing fluorochromes and buffer. As a result, a trimethoxybenzoyl derivative of coumatine phosphate has been proved to give a higher sensitivity than a conventional colorimetric method; it is possible to detect 0.2 pg of nucleic acids with this fluorochromw on nylon membrane. Unfortunately, however, the fluorogenic product has no affinity to nitrocellulose membrane which is commonly used for blot analyses of nucleic acids. We are now attempting to modify this fluorochromw so that it may be much less water-soluble and much more adherent to nitrocellulose membrane. We ara confident that the sensitivity of this new method can be amplified by use of a sensitive photon detector down to a femto gram level. Less
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