Kazuhiko Tsukada

Multiplex short tandem repeat typing in degraded samples using newly designed primers for the TH01, TPOX, CSF1PO, and vWA loci

We performed multiplex polymerase chain reaction (PCR) for the TH01, TPOX, CSF1PO, and vWA loci using a newly designed pair of primers that yield smaller fragments than reported previously [Fujii et al., J Hum Genet 45 (2000) 303; Lederer et al., Int J Legal Med 114 (2000) 87]. These loci can be detected in the range of 74-143 bp amplifying products. This system required genomic DNA in a range of 80 pg to 2 ng, and proved to be a sensitive typing method. We compared our system against the GenePrint Fluorescent STR Multiplex Systems CTTv (Promega, Madison, WI, USA), using DNA extracted from old bloodstains left to stand for 17-26 years at room temperature. With our designed system, all allele-typing efforts were successful in the range of 1-5 ng DNA, while no signal peaks were detected, even with when using 10 ng of DNA GenePrint Fluorescent STR Multiplex Systems CTTv.

Investigation of DNA extraction from hair shafts

Abstract Human hair shafts can be important forensic evidence for identification, but DNA typing, even of mitochondrial DNA (mtDNA), presented certain difficulties. We describe three DNA extraction methods from hair shafts, such as the phenol/chloroform method, NaI treatment method, and silica-beads method. In order to make an investigation of mtDNA amplification rate and efficiency, the amplifications of the mtDNA control region (D-loop) HV1A (15997–16262) used FAM-labeled forward primer. As a result of the extractions from different lengths of fresh hair shafts and the variations of the template volume, fluorescent peak heights as DNA recovery by three methods were sufficiently high. In the degraded sample, a high fluorescent peak height enough to sequence mtDNA could be obtained from our NaI method and silica-beads method.

Molecular genetic analysis of the Am phenotype of the ABO blood group system.

Background and Objectives Many sequences of variants in the ABO blood group system have been analysed, but genetic information is not available on the rare Am phenotype blood group. We isolated the Am phenotype in one family and performed molecular analysis on this allele.

DNA typing using AmpFlSTR Y-filer for very long-term stored specimens.

Aware that long-term storage bloodstain generally reduces numbers of detectable loci, we performed DNA typing with bloodstains stored at room temperature for 22-30years, then extracted DNA from the bloodstains using the AmpFlSTR Y-filer PCR Amplification Kit. We performed electrophoresis with an ABI 310 Genetic Analyzer and determined alleles using GeneMapper ID v3.2 software. Our results suggest that Y-filer finds certain loci more difficult to anneal than others.

Multiplex PCR using newly designed primers for very short fragments of TH01, TPOX, CSF1PO, and vWA loci

We performed multiplex PCR for the TH01, TPOX, CSF1PO, and vWA loci using newly designed pairs of primers that yield smaller fragments than previously reported [Int. J. Leg. Med. 114 (2001) 285; Am. J. Hum. Genet. 55 (1994) 175; Int. J. Leg. Med. 106 (1994) 183.] [1–3]. This system required genomic DNA in a range of 50 pg–2 ng, and proved to be sensitive as a typing method. Furthermore, it was possible to determine the allele types even from 18-year-old bloodstains.

Investigation of ACTBP2 mutations in the Japanese population

In the investigation of ACTBP2 (human beta-actin related pseudogene H-beat-Ac-psi-2) mutation in the Japanese population, 230 meioses were analyzed, and two cases of paternal mutations were observed. Paternity confirmation analyses were carried out using seventeen genetic marker systems including erythrocyte antigens, HLA types, D1S80, and nine STR loci contained in the AmpFlSTR Profiler Kit. Excluding ACTBP2, the paternity probabilities for the two cases were each calculated to be over 99.99%. Genotyping of the ACTBP2 locus was performed using the fluorescence detection method under denaturing conditions on an ABI PRISM 310 Genetic Analyzer. After sequencing analysis using the BigDye terminator method, the paternally originated alleles of the two children were found in one case to show one repeat insertion and in the other case one repeat deletion as compared with each father. Although the number of meioses observed in this study is limited, it appears that ACTBP2 mutations are not rare events in the Japanese population.

Investigation of mtDNA heteroplasmy discordance between mother and child

Abstract Maternal mitochondrial DNA (mtDNA) heteroplasmy discordances were investigated by means of mtDNA hypervariable region 1 sequencing using hair shaft and blood samples obtained from eight mother–offspring pairs. Despite mtDNAs inheritance through the maternal line, heteroplasmic discrepancies appear to exist. Results also suggest that heteroplasmic mutations may be more common in hair than in blood.