Basic Studies on DNA Polymorphism for Application to Foresic Medicine

(1)酶切位点胞嘧啶甲基化对DNA分型的影响研究了内源性5-甲基胞嘧啶对DNA指纹图谱的影响。甲基化敏感的限制性内切酶Sau 3AI和其甲基化不敏感的异构体MboI的分析表明，由于5-甲基胞嘧啶在识别位点产生的模式中存在一些差异。此外，精子DNA的一些条带与来自同一个体的血液DNA的条带不匹配，这种现象仅在甲基化敏感性核酸内切酶中观察到。这些发现表明，甲基化敏感的限制性内切酶的DNA指纹图谱和其他形式的DNA分型的不适合，因为组织特异性的甲基化状态。(2)硬组织DNA提取法鉴定性别--两具未鉴定的漂白骨骸的DNA分析和形态学鉴定 ...更多信息 被执行。骨头和牙齿被取出进行DNA分析，并通过液氮裂解成化合物。通过标准程序提取DNA。我们采用两种DNA分析方法进行性别鉴定，一种是Y染色体特异性探针（PHY 10; 3.4kb）的Southern印迹杂交，另一种是性染色体特异性片段（α随体家族）的PCR扩增。这两种方法使我们能够更准确地确定性别的两个案件在这里提出了充分的证据，而不是仅仅依靠形态学检查。将DNA分析（包括Southern印迹杂交或PCR）应用于硬组织（如一块骨头或牙齿）和旧样本的实际检测中存在一些技术困难。因此，它们似乎是适用于法医学的有效方法。(3)探头行走：新型DNA指纹探针的开发利用人c-Ha-ras-1癌基因下游28个碱基对的串联重复序列作为DNA指纹探针进行了研究。在低严格性下通过Southern杂交观察到多个高变异模式。我们克隆了一个33-bp的串联重复序列，它与28-bp的重复序列交叉杂交。该33-bp重复序列通过Southern杂交在相同严格性下检测到另一组高变限制性片段。这些结果表明，“探针步行”可以用来开发新的探针，提供不同的DNA指纹。少

(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