アブストラクト（43巻2号：神奈川歯学）

神奈川歯学

Japanese

Title　： 混合試料におけるミトコンドリアDNAの検出限界の基礎的研究
Subtitle　： 原著
Authors　： 小林範彦, 大平寛, 山室好生, 山本伊佐夫
Authors（kana）　：
Organization　： 神奈川歯科大学社会医歯学系社会歯科学講座法医学分野
Journal　： 神奈川歯学
Volume　： 43
Number　： 2
Page　： 97-103
Year/Month　： 2008 / 12
Article　： 原著
Publisher　： 神奈川歯科大学学会
Abstract　： 「緒言」 ミトコンドリアDNA(mtDNA)は細胞質に存在する小器官であり, 環状の二重鎖DNAを含んでいる. mtDNAのD-loop領域には塩基置換の蓄積した高度多型領域(HV(Hyper variable)1, HV2, HV3)が存在し1～4), 法医学において多型を利用した個人識別が広く行われており5～12), その他のコード領域においてはPoint Mutation(点変異)が認められ, 疾病との関わりも報告されている13,14). また, 核DNAと比較して, 哺乳動物1細胞あたり10 3から10 4コピー以上存在する15)ことから, 非常に低分子化した試料や極微量の試料からのPCR増幅16)も可能である17～22)と報告されている. さらに, mtDNAは母系遺伝する23～25)ことから, 親子鑑定にも利用されており, 母系をさかのぼることで人類学・遺伝学の分野でも用いられている26,27). 法医学におけるDNA分析は, 扱う試料が微量であり, しかも混合しているものが多く, それらの試料の分析を可能にすることを目標に研究が進められてきた経緯がある.
Practice　： 歯科学
Keywords　： ミトコンドリアDNA, 検出限界, 高度多型領域, 混合試料

English

Title　： Possibility of individual identification from mixed samples using mitochondrial DNA analysis
Subtitle　：
Authors　： Norihiko KOBAYASHI, Hiroshi OHIRA, Yoshio YAMAMURO, Isao YAMAMOTO
Authors（kana）　：
Organization　： Division of Forensic Medicine, Department of Dental Sociology, Kanagawa Dental College
Journal　： Kanagawa Shigaku
Volume　： 43
Number　： 2
Page　： 97-103
Year/Month　： 2008 / 12
Article　： Original article
Publisher　： Kanagawa Odontological Society
Abstract　： [Abstract] Mammalian mitochondrial DNA (mtDNA) is a circular genome approximately 16.5kbp in length that encodes 13 subunits of the inner-membrane respiratory complex. The complete nucleotide sequences for humans have been reported. Non-coding hypervariable segment HV1, the control region, is the most variable region of the human mtDNA. MtDNA shows a maternal mode of inheritance, and the D-loop region is present in sufficiently high amounts to be used for identification casework, since the rate of mutation in mtDNA is 10 times faster than in nuclear DNA. Although mtDNA is reported to be effective for analysis of very small quantities of specimen, its value in mixed specimens is unknown. In this study we examined the efficacy of mtDNA determination in identical mixed specimens, an assignment that is common in DNA analysis in the field of forensic medicine. In the mtDNA HV1 sequence chart from the mixed specimen, overlapping of multiple peaks from different persons was recognized at sites of substitution. However, the correlation between peak heights on the chart and DNA concentration was unclear. Therefore, we first investigated the amplification limit of mtDNA, and then the detection limit of mtDNA in mixed specimens from two samples of DNA. In the sequence chart for mixed specimens, we recognized overlapping of two peaks at sites of substitutions. Finally, at a mixture ratio of 1:0.05, we were able to recognize it as a mixed specimen on the sequence chart. We carried out PCR amplification with DNA over a concentration ranging from 500pg to 0.5pg, and were able to amplify down to 1pg. The cycle sequence reaction requires about 20ng of PCR product, and its volume should be within 12μl. Therefore, we were able to carry out direct sequencing if the concentration of one of the mixed DNAs was higher than 10pg. We were able to recognize a mixed specimen when the mixture ratio was more than 1:0.05. Therefore, theoretically, if more than 0.5pg of another DNA is also present (1/20 of 10pg), it is possible to recognize that a specimen is a mixed one by using the sequence chart.
Practice　： Dentistry
Keywords　：