Emiko Naito

Multiplex amplified product-length polymorphism analysis for rapid detection of human mitochondrial DNA variations

A number of mutations in coding and noncoding regions of mitochondrial DNA (mtDNA) have previously been studied. In the present study, we simultaneously typed six mutation sites in the coding region by use of amplified product‐length polymorphism (APLP) analysis. The mtDNA variations of 2471 individuals from 20 populations of Japanese, Korean, Chinese, and German were examined and classified into 18 haplotypes. Two of these haplotypes, B1 (estimated ancestral haplotype) and C1, were distributed among all populations tested. However, the haplotypes A1, A2, B2, B3, and C2 were mostly restricted to the Mongoloid populations, whereas haplotypes B5 and C5 appeared almost exclusively in the German population. Phylogenetic analysis by the neighbor‐joining method revealed that the Japanese populations were more closely related to each other than to the other East Asian populations surveyed. The multiplex APLP method is suitable for large‐scale screening studies of mtDNA variability because it is both rapid and economical.

Ribosomal Ribonucleic acid (rRNA) gene typing for species identification

Deoxyribonucleic acid (DNA) typing of ribosomal ribonucleic acid (rRNA) genes was performed with a polymerase chain reaction (PCR) assay for species identification. A variable region of the 28S ribosomal RNA gene was amplified with primers complementary to flanking sequences phylogenetically well conserved. The products of twelve animal DNAs (human, Japanese monkey, dog, cattle, pig, cat, rabbit, mouse, rat, chicken, frog, and fish) were separated by polyacrylamide gel electrophoresis, each revealing a few bands ranging from 150 to 100 base pairs. The band patterns obtained from each DNA sample differed in number and size, which indicates the applicability of the method to species identification. Samples containing either as little as 1 pg of DNA or degraded DNA of 0.2 to 0.5 kb in length were able to give detectable bands. Postmortem human tissue DNAs were tested as an example. They showed a pattern identical to the human control one, which was distinct from those of the other animals examined.

Sex Typing of Forensic DNA Samples Using Male- and Female-Specific Probes

Forensic DNA samples have been examined to ascertain the feasibility of a sex-typing procedure that we have recently developed. This uses two sets of primers complementary to the DXZ4 and SRY genes for polymerase chain reaction (PCR). PCR target in the DXZ4, an 80-bp sequence within the 130-bp fragment specific to females, is generated from inactive chromosome X by the DNA digestion with a methylation-sensitive restriction enzyme, HpaII. Therefore, the DXZ4 amplification and subsequent agarose gel electrophoresis detect the 80-bp fragment from female DNA. On the other hand, the SRY probe identifies a male-specific sequence on chromosome Y. Testing DNAs from fresh Turners blood and from postmortem tissues exhibited band-signals confirming the sex identification. Degraded DNAs isolated from severely decomposed specimens were also identifiable when high-molecular-weight DNA was isolated before the assay. This demonstrates the usefulness of this method in forensic identification.

A novel dimorphism in the human SRY gene: usefulness in human migration studies.

Abstract A nucleotide polymorphism of C or T was detected at position 465 in the sex-determining region Y (SRY) gene. To evaluate the utility of this dimorphism in human population studies, the frequency and the frequency of the haplotype combined with the two polymorphic loci YAP and M9 were examined in a total of 130 unrelated Japanese and 130 unrelated German males. The T nucleotide was found in 24.6% (32/130) of the Japanese but not in any of the 130 German males. Accordingly, four of the eight possible combination haplotypes of SRY/YAP/M9 were identified in the Japanese population, but one of the four haplotypes comprising SRY(T) was absent in the German samples. This suggests that the C to T transition may be more recent than the YAP insertion or the M9 transversion and the change might have occurred in an ancestral Asian population. These results imply that the dimorphism at the SRY gene is one of the Y-linked markers useful for human population studies and also for ethnic identification of forensic samples.

A VNTR polymorphism in human 5′ H19 flanking regions in Japanese and German populations

In mammals, imprinted genes are preferentially expressed from either the maternal or paternal allele. Several recent observations show that DNA methylation plays an important role in the imprinted inheritance of the gene. Generally, an individual inherits two alleles together from his parents and the origin of each allele can be determined by typing the parents. The ultimate aim of this study is to detect a paternally or maternally derived allele from one person by using the methylation difference in the imprinted region. For the purpose, a useful probe within the region is requisite as the analytic target. In this study, the forensic utility of the VNTR locus, which is located approximately 7.6 kb upstream of the H19 gene that is maternally expressed, was evaluated. The human 5VH19 flanking sequence was searched from the DDBJ. The allele frequency of the VNTR locus was examined in a total of 199 unrelated Japanese and 171 unrelated German individuals. In the Japanese samples, 7 alleles and 22 genotypes were identified. The heterozygosity and polymorphism information content (PIC) were 0.749 and 0.669, respectively. By contrast, 9 alleles and 27 genotypes were detected in the German samples. The heterozygosity and PIC were 0.969 and 0.705, respectively. Thus, the frequency distribution of the two populations showed different profiles (Table 1). In a Japanese case study, this genetic typing was successfully applied to the personal identification of decomposed remains as a forensic sample. These results indicated that the VNTR in the H19-5Vflanking region is highly polymorphic and useful for personal identification of both Japanese and German DNA samples. This suggests the VNTR has become a useful probe for the aimed method.