The polymorphism of HLA-DMB gene and its application to the personal identification.

HLA-DMB基因多态性及其在个人身份识别中的应用

• 批准号: 11670407
• 负责人: SATO Yayoi
• 金额: $ 1.98万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670407/
• 关键词: DNA; HLA-DMB; PCR-SSCP; polymorphism; PCR-SSP; Hot Start PCR; Forensic Science; PCR-RFLP; Direct Sequencing; polvmorphism

HLA-DMB typing was performed from forensic samples (blood, bloodstain, hair, saliva stain, cerebral dura mater, urine, bone and tooth) using PCR-RFLP method. PCR products of the DMB allelic third exon was digested with three restriction endonucleases such as ApaL I, HinPl I, Bsr I.After digestion, samples were electrophoresed in 12% acrylamide gels. Most PCR products could be digested by those endonucleases and eight generic types were observed. Ninety-two percent of all the sample (N=129) was identified and eighty-three percent of the hair sample was identified. DMB^*0106 allele were not identified in this study. Although three pairs of heterozygous generic types could not distinguished each other, they could be typed by PCR-SSP method. DNA typing for HLA-DMB gene by this method could be available for the forensic applications such as personal identification, because of its reproducibility and simplicity.DNA typing for DMB using PCR-SSP method was also studied. The problem of the false positive and false negative should be resolved. The screening of the allelic variations was also studied by PCR-SSCP method and two types were observed in the first exon. About another exon the screening will be performed and direct sequencing of the PCR products will be analyzed. The differentiation of the allelic variations was difficult by PCR-SSCP method and the primer design and the strategy should be reconsidered.In this study, sample DNA was too little to analyze the DMB allelic sequence using direct sequencing method.

采用PCR-RFLP法对法医标本（血液、血迹、毛发、唾液、脑膜、尿液、骨骼和牙齿）进行HLA-DMB分型。DMB等位基因第三外显子PCR产物用ApaL I、HinPl I、Bsr I三种限制性内切酶酶切，酶切后用12%丙烯酰胺凝胶电泳。大多数PCR产物能被这些内切酶消化，并观察到8种类型。92%的样本（N=129）被鉴定，83%的头发样本被鉴定。本研究未发现DMB^*0106等位基因。虽然三对杂合型不能相互区分，但可以用PCR-SSP方法进行分型。该方法具有重复性好、操作简单等优点，可用于个人鉴定等法医鉴定。采用PCR-SSP方法对DMB进行了DNA分型研究。应该解决假阳性和假阴性的问题。用PCR-SSCP方法筛选等位基因变异，在第一外显子中发现两种类型。对另一个外显子进行筛选，并对PCR产物进行直接测序分析。PCR-SSCP方法难以区分等位基因变异，引物设计和策略需要重新考虑。在本研究中，样品DNA太少，无法使用直接测序法分析DMB等位基因序列。