Sudy on the heteroplasmic substitutions in the mitochondrial DNA control region

线粒体DNA控制区异质取代的研究

• 批准号: 13670419
• 负责人: FUKUSHIMA Hirofumi
• 金额: $ 0.77万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670419/
• 关键词: PCR; subtyping; heteroplasmy; polymorphism; D loop; hair shafts; sequence; variants; HVI; HVII

Detection of mitochondrial DNA variation is commonly carried out in Forensic Science. The control region of mtDNA is highly polymorphic and useful for individual identification purposes.Alternative strategies to DNA sequencing have been proposed for the detection of mutation. For the screening of mtDNA heteroplasmy, SSCP may be the most utilized method. This method is based on the different conformations which single stranded DNA adopts. Our results with 100 unrelated individuals showed that using this method one individual has differents patterns.We described three methods for extracting DNA from hair shafts, including the Phenol / Chloroform method, the NaI treatment method, and the Silica - beads method. With the degraded sample, amplification from the template by the Phenol / Chloroform method produced no results. In contrast, high fluorescent peak heights adequate for sequencing mtDNA were obtained from our two methods, confirming that the NaI treatment and Silica - beads methods are volid, reliable means of extracting and amplifying mtDNA from hair shafts.The sequences of the HV1 and HV2 regions from close maternal relatives (mother - child pairs) were compared to determine the frequency of heteroplasmic mutations. A total of 16 blood and hair samples were sequenced. The several heteroplasmic point mutations were found in mother and child samples.

线粒体DNA变异检测是法医学中的一项重要工作。线粒体DNA控制区具有高度多态性，可用于个体识别。SSCP可能是线粒体DNA异质性筛选中最常用的方法。该方法基于单链DNA所采用的不同构象。本文介绍了三种从毛干中提取DNA的方法，即苯酚/氯仿法、碘化钠处理法和硅珠法。对于降解样品，通过苯酚/氯仿方法从模板扩增未产生结果。相比之下，我们的两种方法都获得了足以测序mtDNA的高荧光峰高度，证实了NaI处理和硅胶珠法是从毛干中提取和扩增mtDNA的有效、可靠的方法。共对16份血液和头发样本进行了测序。在母亲和儿童样本中发现了几个异质性点突变。

项目成果

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Asamura H, Takayanagi K, Tsukada K, Ota M, Fukushima H.: "DNA analysis of the two pieces of bone founded in the mountains."DNA polymorphism. 11. 271-273 (2002)

Asamura H、Takayanagi K、Tsukada K、Ota M、Fukushima H.：“在山上发现的两块骨头的 DNA 分析。”DNA 多态性。

Asamura H., Takayanagi K., Ota M., Kurita H., Kurashina K., Fukushima H.: "Personal Identification by Mitochondrial DNA Control Region: Cases of Severely Damaged Cadaver Lacking Dental and Fingerprint Information."Res Pract Forens Med. 45. 157-161 (2002)

Asamura H.、Takayanagi K.、Ota M.、Kurita H.、Kurashina K.、Fukushima H.：“通过线粒体 DNA 控制区域进行个人识别：缺乏牙齿和指纹信息的严重受损尸体案例。”Res Pract Forens Med。

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Takayanagi K., Asamura H., Tsukada K., Ota M., Fukushima H.: "Hair analysis for human and species identification."DNA polymorphism. in press (2003)

Takayanagi K.、Asamura H.、Tsukada K.、Ota M.、Fukushima H.：“用于人类和物种鉴定的头发分析。”DNA 多态性。