Kayoko Takayanagi

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.

Allele frequencies of the six miniSTR loci in a population from Japan.

Allele frequencies and forensic parameters for the six miniSTR loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434, and D22S1045 were investigated in a sample of 142 unrelated healthy Japanese individuals. The polymerase chain reaction (PCR) products contained within the six loci were less than 119 bp in size. The frequency distributions in the six short tandem repeat (STR) loci showed no deviations from Hardy–Weinberg equilibrium expectations. The accumulated powers of discrimination and power of exclusion for the six loci were 0.999998 and 0.98, respectively. It was thus considered that due to the small PCR products and the moderate degree of polymorphism, analysis with use of the six miniSTR loci was highly beneficial for the forensic analysis of degraded DNA.

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.

D1S80 subtyping by PCR-RFLP: new nomenclature and further characterization.

In a previous study, we screened out sequence variations within alleles at the D1S80 locus of a Japanese population using PCR-RFLP with EcoRII as a restriction enzyme. In the present study, through analyzing the alleles in a Chinese population, we were able to demonstrate four new electrophoretic band patterns that were complementary to the Japanese data. After summarizing the band patterns and sequencing results of these two populations, we established a new nomenclature for the PCR-RFLP band patterns, closely relating them to their corresponding sequences so that the new types could be designated easily and accurately. After PCR-RFLP subtyping, nineteen alleles in the Chinese population were revealed to have a total of thirty-seven subtypes. The discrimination power of this locus in the Chinese population was elevated from 0.974 to 0.988, and the Hardy-Weinberg equilibrium in this population showed no deviation when checked. Two samples typed as homozygotes 24/24 and 30/30 were identified to be actually heterozygous according to their band patterns. The result was supported by the sequencing analysis of the two samples in which overlapping of eight and eleven repeat units, respectively, were revealed. The heterozygosity was thus elevated from 0.85 to 0.87. The present study proved that PCR-RFLP was an effective method for subtyping D1S80 alleles in the Chinese.

Application of short tandem repeat of genomic DNA and mitochondrial DNA for identification of mixed-up tissue specimens

Deoxyribonucleic acid (DNA) typing methods, short tandem repeat of genomic DNA and mitochondrial DNA with the use of polymerase chain reaction amplification, were applied to formalin‐fixed, paraffin‐embedded tissues submitted for diagnosis, to identify and sort out mixed‐up tissue specimens. These techniques were found to be reliable, reproducible and specific for personal identification, and thus to eliminate the need for further examinations and to prevent unnecessary surgery.

The judgment of a gunshot wound with severe post-mortem changes

This is a report on an autopsy case that revealed a gunshot wound to the back of the head. On examination of the external surface, an entrance and exit wound could not be found in the head region because of the severe post-mortem changes and the atypical entrance wound position and direction of the bullet tract; X-rays taken before the internal examination were effective in this case. In this case, the direction of the bullet tract was supposed to be similar to that of the suboccipital puncture.

Case of shaken baby syndrome in Japan caused by shaking alone

(SBS) has gained wide attention since it was first defined by Caffey as ‘whiplash shaken infant syndrome’.1 To date, this syndrome has not been widely recognized in Japan. SBS has generally been defined as intracranial injury caused by shaking in infants 2 years old or less. When first described, SBS was believed to be the result of subdural hematoma caused by bridging-vein collapse due to repeated acceleration/ deceleration movements of the head in vigorous manual shaking.1,2 However, this hypothesis has recently been disputed and it is now generally assumed that SBS occurs through a combination of shaking and direct external force to the head.3,4 In the present report we describe an autopsy case of a 3-month-old male infant where the cause of death appeared to be attributable to SBS from shaking alone, occurring in the course of playing with the infant without malicious intent.

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.