| Project/Area Number |
01480212
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Legal medicine
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| Research Institution | University of Tsukuba |
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Principal Investigator |
MISAWA Shogo Univ. of Tsukuba, Inst. Community Med. Sci., Prof., 社会医学系, 教授 (50086534)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Einosuke Univ. of Tsukuba, Inst. Community Med. Sci., Assist. Prof., 社会医学系, 講師 (30138416)
HARADA Shoji Univ. of Tsukuba, Inst. Community Med. Sci., Associate Prof., 社会医学系, 助教授 (60086618)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1990: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1989: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | DNA Polymorphism / DNA Fingerprint / Probe / RFLPs / Southern hybridization / Mitochondrial DNA / PCR / VNTR / RFLPs(制限酵素断片長多型) / サザンプロッテイング / PCR法 / 制限酵素 / サザンハイブリダイゼイション / Ha-ras遺伝子 / 個人性別 / 親子鑑定 |
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| Research Abstract |
(1) Effects of Cytosine Methylation at Restriction Sites on Deoxyribonucleic Acid (DNA) Typing
The effects of endogenous 5-mrthylcytosines on deoxyribonucleic acid (DNA) fingerprints were studied. Analysis with methylation-sensitive restriction endonuclease Sau3AI and its methylation-insensitive isoschizomer MboI showed some differences in the patterns generated as a result of 5-methylcytosines at the recognition sites. Moreover a few bands of sperm DNA did not match those of blood DNA from the same individual, a phenomenon only observed in the digests of methylation-sensitive endonucleases. These findings indicate the unsuitability of methylation-sensitive restriction endonucleases for DNA fingerprinting and other forms of DNA typing, because of tissue-specific status of the methylation.
(2) Sex Identification by Analysis of DNA Extracted from Hard Tissues
Sex identification of unidentified two bleached skeletons by the method of DNA analysis in addition to the morphological examination
… More
was performed. Bones and teeth were taken out for the DNA analysis, and cracked to compounds by liquid nitrogen. DNA was extracted by the standard procedure. We used two methods for sex identification by DNA analysis ; one is Southern blot hybridization with Y-chromosome specific probe (PHY10 ; 3.4kb), and the other is PCR (polymerase chain reaction) amplifying with sex chromosome specific fragments (alphoid satellite family). These two methods enable us to identify the sex of the two cases presented here more correctly with sufficient evidence than depending only on morphological examination. With some technical difficulties, it is possible to put DNA analysis including Southern blot hybridization or PCR into practical determination of seing of hard tissues such as a piece of bone or a tooth, and old samples. Therefore, they seem to be effective methods applicable to forensic science.
(3) Probe walking : development of novel probes for DNA fingerprinting
The tanderm repeat of a 28-base-pair (bp) sequence downstream of the human c-Ha-ras-1 oncogene was studied as a probe for DNA fingerprinting. Multiple hypervar able patterns were observed by Southern hybridization at low stringency. The patterns were specific to individuals, we cloned the tandem repeat of a 33-bp sequence, which cross-hybridized with the 28-bp repeat. This 33-bp repeat detected another set of hypervariable restriction fragments by Southern hybridization at the same stringency. These results suggests that "prob walking" can be employed to develop novel probes that provide different DNA fingerprints. Less
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