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Development and Practical Use of Highly Sensitive Detection Methods for Individualization Markers

个体化标记高灵敏检测方法的开发和实际应用

基本信息

批准号：
12357003
负责人：
YASUDA Toshihiro
金额：
$ 22.72万
依托单位：
Faculty of Medical Sciences, University of Fukui
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

项目摘要

1.We have previously found human deoxyribonuclease(DNase) II to be a genetic marker : DNase II levels in human vary depending on whether the individual has the DNA SF2 *H or *L allele.It was clarified that the A-75G transition in the proximal promoter region causes the allelic difference in the promoter activity of the gene, underlying its genetic polymorphism.2.We devised a simple DNA extraction procedure suitable for STR typing of urine samples using a commercially available DNA/RNA extraction kit. This method alows urine samples to be kept in both a frozen or aqueous state for forensic use.Furthermore, we devised a procedure that combines a simple extraction method, isoelectric focusing and activity-staining for DNase I typing from aged urine stains.DNase I polymorphism could be considered the first biochemical marker found to be well suited for individualization from small aged urine stains.3.We confirmed the population genetic basis for DNase I polymorphism in a Japanese populatio … More n for forensic application. Our examination of DNase I types revealed a decreasing north-to-south gradient in the DNA SE1 allele.4.We discovered a novel variable number of tandem repeat of 56 bp unit in intron 4 of human DNase I gene, resulting in elevation of individualization efficiency of DNase.5.In order to devise the highly sensitive-and specific-immunological typing for DNase I and II polymorphisms, we succeeded in production of each murine monoclonal anti-human DNase I and DNase II with high specificity using newly developed screening procedure for antibodies.Immunoaffinity procedure using these monoclonal antibodies made the purification of human DNases I and II easier, faster and more effective than the conventional procedure. Furthermore, in order to obtain human DNase I as an immunogen, we developed a procedure consisting of transient expression of the enzyme in transfected mammalian cells with DNase I cDNA and purification of the enzyme from the culture medium.6.Molecular-evolutional aspects of vertebrate DNase I family have been clarified. Less

1.我们以前发现人类脱氧核糖核酸酶（DNase）II是一种遗传标记：人的DNA酶II水平根据个体是否具有DNA SF 2 *H或 *L等位基因而变化。已阐明近端启动子区域中的A-75 G转换导致基因启动子活性的等位基因差异，2.我们设计了一个简单的DNA提取程序，适用于使用市售的DNA/RNA提取试剂盒对尿液样本进行STR分型。该方法允许尿液样本以冷冻或含水状态保存以供法医使用。此外，我们设计了一种结合简单提取方法，等电聚焦和活动-结果表明，老年尿斑DNA酶I多态性是老年尿斑个体化的第一个生化标志物。3.我们证实了老年尿斑DNA酶I多态性的群体遗传基础，日本人群DNA酶I多态性 ...更多信息 n用于法医应用。4.在人DNase Ⅰ基因内含子4中发现了一个新的可变数目的56 bp串联重复序列，它能提高DNase的个体化效率。5.为了设计高灵敏度和高特异性的DNase Ⅰ和DNase Ⅱ多态性免疫分型方法，我们用新开发的抗体筛选方法成功地制备了特异性高的鼠抗人DNase Ⅰ和DNase Ⅱ单克隆抗体，用这些单克隆抗体进行免疫亲和纯化，使人DNase Ⅰ和DNase Ⅱ的纯化比常规方法更简便、快速和有效。此外，为了获得作为免疫原的人DNA酶I，我们建立了一种方法，包括在转染DNA酶I cDNA的哺乳动物细胞中瞬时表达该酶，并从培养基中纯化该酶。6.阐明了脊椎动物DNA酶I家族的分子进化方面。少