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

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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