Basic Studies on DNA Polymorphism for Application to Foresic Medicine

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(1) Effects of Cytosine Methylation at Restriction Sites on Deoxyribonucleic Acid (DNA) TypingThe 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 TissuesSex 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 fingerprintingThe 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