Short tandem repeat typing of cells transferred via micromanipulation with whole-genome amplification.

This study attempted to determine the minimum number of cells required to conduct DNA analyses effectively. Oral mucosal cells obtained from eight persons were suspended and individually collected by using micromanipulation technique. DNA was extracted and amplified by whole-genome amplification (WGA). Nuclear DNA was extracted to evaluate the feasibility of autosomal short tandem repeat (STR) polymorphism and Y-chromosomal STR polymorphism analyses. Tests were conducted with 20 and 30 cells, to determine the minimum number of cells required for each DNA analysis. Tests with 20 cells were repeated 5 times, to examine reproducibility. When five or 10 cells were used, loci could not be identified for most alleles. Furthermore, DNA polymorphism analyses of a single cell transferred directly to a polymerase chain reaction solution were unsuccessful. The present findings suggest that, in forensic identification, 20 or more cells are required in order to obtain clear results from autosomal and Y-chromosomal STR polymorphism analyses. Furthermore, the feasibility of sample preservation and reexamination was also confirmed by DNA amplification with WGA.

Social contribution of forensic odontology in Japan.

Half a century has passed since the department for education and research on forensic odontology was established at dentistry-related universities in Japan in 1964. In order to meet the demands of society, the number of universities with a department of forensic odontology increased up until around 2005. In 2007, the Japanese Society of Forensic Dental Science was established, and then a series of reforms such as establishment of the Study Council on Death Cause Investigation in both the National Police Agency and the Cabinet Office of the Japanese government, cabinet decision of enactment and enforcement of new laws on death cause investigation, publication of an article on the Model Core Curriculum of Dental Education, publication of the results of a fact-finding survey on education and research on forensic odontology conducted by the Ministry of Education, Culture, Sports, Science and Technology, inclusion of questions about forensic odontology in the National Board Dental Examination, and compilation of a database on dental findings by the Ministry of Health, Labor and Welfare, proceeded in succession. We introduced the half century of forensic odontology in Japan in chronological order.

DNA analysis of root canal-filled teeth.

Teeth are markedly useful as samples for DNA analysis; however, intact teeth are not always available. This study examined the possibility of identifying autosomal and Y-chromosome short tandem repeat (STR) types in samples from 34 teeth (15 intact and 19 root canal filled) that had been preserved for 10-33years after dental extraction. The aim was to explore the feasibility of individual identification by DNA analysis of samples obtained from highly decomposed and skeletonized corpses. Only one out of 24 autosomal STR loci was not identified in two of the 15 intact teeth, whereas all 23 loci of the Y chromosome STR were detected. One or two autosomal STR loci remained unidentified in eight of the 19 root-filled teeth, and four or five of the 23 Y STR loci were undetected in three cases. However, the types were identified in about 20 loci in all samples, and the composition of the root canal filling material did not appear to interfere with the PCR. This study demonstrates that the storage period of the teeth had no influence on our results indicating that root canal filled teeth can be used for DNA analysis.

Analysis of human mitochondrial DNA polymorphisms in the Japanese population.

The highly polymorphic nature and high amplification efficiency of mitochondrial DNA (mtDNA) is valuable for the analysis of biological evidence in forensic casework, such as the identification of individuals and assignment of race/ethnicity. To be useful, a mtDNA polymorphism database for the Japanese population requires an understanding of the range of haplotype variation and phylogenies of mtDNA sequences. To extend current knowledge on the haplotypes in the Japanese population, this study defines new lineages and provides more detail about some of those previously described. We compared the hypervariable regions (HVRs) of 270 healthy, unrelated Japanese individuals and demonstrated 192 haplotypes. Combining HVR1 and HVR2, the genetic diversity was 0.9935, thus providing a high level of identification capability. Haplogroup status was defined for 160 individuals using HVR1, HVR2, and particular coding region polymorphisms; these individuals belonged to 94 haplotypes, four of which were new lineages. The complete mtDNA sequence was also determined from seven individuals.

ABO blood group genotyping by quenching probe method.

We developed a multiplex ABO genotyping method with quenching probes (Q-probe). In this method, it is possible to discriminate the mutations, not only frequently used positions 261 and 796 but also position 703 in a single PCR. Each probe was designed to have cytosine residue at 5' or 3' end and labeled with three different fluorescence dyes, enabling the triplex detections of these polymorphisms. All polymorphisms were successfully detected by using fluorescence labeled Q-probe in a specifically amplified PCR product. Each Q-probe showed unique dissociation patterns depending on the polymorphism types. All of the results obtained with Q-probe were compared with standard serotyping and TaqMan PCR method and resulted in complete match with each other. Consequently, these results indicated that multiplex ABO genotyping method is quite accurate and convenient method for the determination of ABO genotype.

A case of personal identification due to detection of rare DNA types from seminal stain.

Following a rape incident in an apartment in Japan, we were requested to perform a DNA analysis on a body fluid stain left on a bath towel to determine whether it could be attributed to the suspect. The acid phosphatase and prostatic-specific antigen tests confirmed it to be a seminal stain. Based on the DNA analysis by autosomal and Y-chromosome short tandem repeat (STR) systems, no inconsistency was found with the profile of the suspect with African ancestry. In this case, allele 21 of DYS390 at the Y-STR locus was examined, as it is reported to have a distinctly lower frequency in the Japanese population. Furthermore, the haplotype combinations of Y-STR at the DYS389I, DYS389II and DYS390 loci are powerful for personal identification, as these have not yet been found in the Japanese population.

Genetic studies of eight X-STRs in a Japanese population.

We studied eight X-STRs (DXS7132, DXS7423, DXS8378, DXS10074, DXS10101, DXS10134, DXS10135, HPRTB) polymorphism in 494 unrelated Japanese individuals (313 males, 181 females) using Mentype Argus X-8 PCR Amplification Kit. PD of the eight X-STRs ranged from 0.558 (male) to 0.987 (female). Allele frequencies, number of alleles, and PIC were 0.001-0.587, 6-20, and 0.470-0.913, respectively.

Rapid and simple sex determination method from dental pulp by loop-mediated isothermal amplification.

Sex determination from dental pulp DNA was examined by loop-mediated isothermal amplification (LAMP) method. Amelogenin locus was analyzed for sex determination. A set of four specially designed primers was prepared based on database from Gene Bank, and loop primers were designed to shorten the analysis time. Analysis was performed using 32 dental pulp DNA samples removal from permanent teeth stored at room temperature for 1-25 years after extraction. The X allele was detected in approximately 32min with real-time turbidimeter and the Y allele was detected in approximately 34min. Analysis time was reduced to half when using loop primers. Visual detection was also possible as the amplified product showed white turbidity. Sex determination by LAMP method was rapid and simple, and it should prove useful in unknown bodies of mass disasters.

Polymorphism of the vWA locus located in the center of the three vWA STRs in the Japanese population.

The structural polymorphism of the vWA locus (vWA-T) located between the two polymorphic vWA loci (vWA-K and -P) was analyzed in 100 Japanese individuals using DNA samples isolated from dental pulp. The polymorphism of this locus was based on the difference in the number of tcta repeat. New interallele 11.1 was found in two samples. All together 9 alleles and 19 genotypes were observed. In addition, one mutant allele contained tcga in the common tcta repeat structure. The value of PD was calculated to be 0.900. Inheritance of the polymorphism was confirmed in a family including 23 individuals and 6 matings.

Hypervariable region structure and polymorphism of mtDNA from dental pulp and a family analysis.

Nucleotide sequences of the hypervariable region in the D-loop of mitochondrial DNA (mtDNA) were analyzed using DNA extracted from 140 old dental pulp samples. These sequences were compared with the sequence reported by Anderson et al. Nucleotide substitution in the HV1 region was identified at 77 positions. A C-to-T transition at position 16223 (C16223T) was most frequently detected (77.9%). Fourteen types of C-stretch sequence patterns were detected and the same sequence as Anderson had the highest frequency (57.9%). In the HV2 region, base transitions were identified at 56 positions. A263G was identified in all samples. Seven types of C-stretch were detected, but none had the same sequence as Anderson. In the HV3 region, base transitions were identified at 21 positions. T489C was most frequently identified (64.3%). Five types of C-stretch were detected, and the same sequence as Anderson accounted for 92.9%. The 140 samples were classified into 128 kinds by the sequence patterns of the HV region. Next, using the blood and oral mucosa epithelium from 23 subjects comprising four generations in a family line, the hereditary relationship of mtDNA was examined. All mtDNA types of the first-generation mother were infallibly inherited by the fourth generation.