The development of a new HLA-DNA typing method using DNA direct sequencing.

使用 DNA 直接测序开发新的 HLA-DNA 分型方法。

• 批准号: 04557031
• 负责人: KATSUMATA Yoshinao
• 金额: $ 3.78万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04557031/
• 关键词: HLA; PCR; Direct Sequence; Forensic Specimens; 法医鑑識; 親子鑑定; 犯罪捜査; 白血球型; 血液型; 遺伝子増幅; 塩基配列直接解読; 蛍光標識プローブ

Highly polymorphic human leukocyte antigen (HLA), which is related to cellular immunoreaction, is useful for personal identification in the field of forensic medicine. To date, HLA class II loci are typed by restriction enzymes or sequence-specific oligonucleotide probes after being amplified by the polymerase chain reaction (PCR). Such procedures are usually complicated and time consuming. Therefore, direct sequencing of the amplified DNA could provide a rapid typing method. However, forensic specimens such as bloodstains, seminal stains, saliva stains and hairs usually contains contaminants which may inhibit PCR reaction. In this study, we tried to analyze the nature of the contaminants, and to minimize the effect of those substances. In hair, we found that melanin is a potent inhibitor for the PCR reaction, and are now trying to minimize the effect of this substances. In bloodstains and saliva stains, we tried to control the inhibition of the contaminated substances, and finally made an improved procedure of DNA purification and typing.So, we started to read the sequence of HLA-DQB1 gene with DNA sequencer.

高度多态性人类白细胞抗原（HLA）与细胞免疫反应有关，在法医学领域有重要的个人识别价值。迄今为止，HLA II类基因座在通过聚合酶链反应（PCR）扩增后通过限制酶或序列特异性寡核苷酸探针分型。这些程序通常复杂而费时。因此，扩增DNA的直接测序可以提供快速分型方法。然而，血迹、精斑、唾液斑和毛发等法医标本通常含有可能抑制PCR反应的污染物。在这项研究中，我们试图分析污染物的性质，并尽量减少这些物质的影响。在头发中，我们发现黑色素是PCR反应的有效抑制剂，现在正试图将这种物质的影响降到最低。在血迹和唾液斑中，我们尝试控制污染物的抑制作用，并最终改进了DNA纯化和分型的程序，从而开始用DNA测序仪读取HLA-DQB 1基因序列。

项目成果

Rieko Uchihi, Keiji Tamaki, Toshinori Kojima, Toshimichi Yamamoto and Yoshinao Katsumata: "Deoxytibonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs in Japanese." Journal of Forensic Sciences. 37(3). 853-859 (1992)

Rieko Uchihi、Keiji Tamaki、Toshinori Kojima、Toshimichi Yamamoto 和 Yoshinao Katsumata：“日本单根毛发中人类白细胞抗原 (HLA)-DQA1 的脱氧核糖核酸 (DNA) 分型。”

Okajima,H.et al.: "Amplification of HLA-DQA1 gene from bloodstains by polymerase chain reaction." Japanese Journal of Legal Medicine. 47(1). 6-12 (1993)

Okajima,H.et al.：“通过聚合酶链式反应从血迹中扩增 HLA-DQA1 基因。”

玉木 敬二: "MUR-PCR法を用いた日本人におけるDIS8(MS32)のタイピングとアリルの解析" 日本法医学雑誌. 46. 474-482 (1992)

Keiji Tamaki：“使用 MUR-PCR 方法对日语中的 DIS8 (MS32) 进行分型和等位基因分析”《日本法医学杂志》46. 474-482 (1992)。

Uchihi,R.et al.: "Deoxyribonucleic acid(DNA)typing of human leukocyte antigen(HLA)-DQA1 from single hairs in Japanese." Journal of Forensic Sciences. 37(3). 853-859 (1992)

Uchihi,R.et al.：“从日本人的单根毛发中对人类白细胞抗原 (HLA)-DQA1 进行脱氧核糖核酸 (DNA) 分型。”

Keiji Tamaki, Rieko Uchihi, and Yoshinao Katsumata: "Biotechnology in medicine(17)-Personal identification by DNA-" Gendaiigaku. 40(1). 169-174 (1992)

Keiji Tamaki、Rieko Uchihi 和 Yoshinao Katsumata：“医学生物技术（17）-通过 DNA 进行个人识别-”Gendaiigaku。