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A procedure for the analysis of mitochondrial DNA (mtDNA) using capillary electrophoresis instead of former gel-based methods is described. The procedure requires less manual manipulation in terms of electrophoresis and, therefore, reduces the chance of either human- or gel-related failures. Thus, the method is suitable for performing mtDNA typing from limited amounts of forensic samples. We also performed mtDNA typing of hair samples using this method, and were successful in typing from both hair roots and hair shafts as well as saliva and nail samples. The amount of PCR product indicated that the amount of mtDNA in the tip side of the hair shaft was less than that in the root side. However, one hair sample showed equal amounts of PCR products in both the tip and the root side. For the analysis of a sample derived from an individual with heteroplasmic mtDNA, the proportions of heteroplasmy from the saliva and nail samples were different from those from hair samples. For analyses of hairs from the same individual, each region of the hair showed different proportions of heteroplasmy and the results indicated the possibility of the different sequences in the same hair sample. Therefore, mtDNA analysis of hair samples will require additional investigation of procedures for heteroplasmic mtDNA. These results strongly suggest that the application of the developed method for hair samples will require careful treatment of the samples and a rigorous analysis of the results.

We describe a new procedure for the multiplex PCR amplification of the three short tandem repeat (STR) loci; TH01, CSF1PO and TPOX with small amplicon lengths (the Small TCT Multiplex). The accuracy of the Small TCT Multiplex was verified by typing 100 Japanese samples that had been previously typed using an AmpFlSTR Green I PCR amplification kit (Green I Kit). The results using the Small TCT Multiplex were consistent with those obtained by the Green I Kit. STR typing using the Small TCT Multiplex was examined from 36 bloodstain samples that had been left for 1 to 25 years at room temperature and compared to that using the Green I Kit. The Small TCT Multiplex was superior to the Green I Kit for STR typing especially in the case of bloodstain samples that had aged for more than 8 years.

Six short tandem repeat loci (DYS19, DYS388, DYS390, DYS391, DS392 and DYS393) on the nonrecombining portion of the human Y chromosome were examined to investigate the usefulness of Y-chromosome haplotyping for human identification purposes in the Japanese and Taiwanese populations. In addition to STR loci, a Y-chromosome Alu insertion polymorphism (YAP) was tested in the Japanese samples.

Three Kinds of DNA analyses; AmpliType® PM typing, TH01 typing and mitochondrial DNA (mtDNA) analysis were evaluated to establish the corroborative method for forensic hair comparison. Five scalp telogen hairs were collected from 12 Japanese males ranging in age from 26 to 33 years. DNA was extracted from five hair shafts of 5cm in length and from five hair roots of 3mm in length taken from each subject. The PM typing was performed using the AmpliType® PM PCR Amplification and Typing Kit. The TH01 typing was carried out using Quick-TypeTM HUMTH01 and was detected by silver staining. For the mtDNA analysis, the sequencings of the two hypervariable regions (HV1 and HV2) of control region were performed by using the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA polymerase FS and ABI PRISMTM model 377 DNA sequencer. In the telogen hair, the hair root was a suitable sample for both PM and TH01 typing compared to the hair shaft samples (about 50% of the subjects could be detected from the hair root samples). The PM typing and TH01 typing showed almost equal detectability. In the mtDNA analysis, the PCR amplifications of HV1 and HV2 were successfully performed in all twelve subjects by employing new primer, and the sequences of the PCR products from two subjects were determined. This result suggests that the mtDNA analysis can be applied for hair comparison in cases where the genomic DNA typing is not available. However, further study for the reliability and reproducibility of the mtDNA analysis should be performed.

Nucleotide sequences of 2 hypervariable regions (HV1, HV2) within the control region of human mitochondrial DNA (mtDNA) were analyzed from 55 unrelated Japanese. About 700 nucleotides were sequenced by using the nested PCR and the solid-phase direct sequencing methods. Comparison of these sequences with Anderson's reference sequence revealed 97 mutation types within 93 positions, and 11 positions of them were novel. Fifty five samples analyzed were classified into 52 different sequences, while 3 pairs have shown the same sequences. Comparison of the Japanese sequences to those reported from other populations indicated many differences in such a point that the substitutions at 16,223 and 73 in Japanese were more frequent than those in Caucasian, while the substitutions at 16,126 and 16,311 in Japanese were less frequent than those in Caucasian. Twenty one of 55 samples analyzed showed a T-to-C transition at the position 16,189 of the C-stretch region in the HV1 region. This replacement caused the blurred bands on the sequence image, which resulted in the ambiguity of exact number of cytosine in the C-stretch region of HV1. For this ambiguity, the number of cytosine in the C-stretch region should not be currently taken into account in forensic practices of individualization of evidence samples. Regardless of such problem, the polymorphisms of HV1 and HV2 regions are highly useful for individual identification.