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We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

Allele frequencies for 31 autosomal short tandem repeat (STR) loci (CSF1PO, D10S1248, D12ATA63, D12S391, D13S317, D14S1434, D16S539, D18S51, D19S433, D1S1656, D1S1677, D21S11, D22S1045, D2S1338, D2S1776, D2S441, D3S1358, D3S4529, D4S2408, D5S2800, D5S818, D6S1043, D6S474, D7S820, D8S1179, FGA, Penta D, Penta E, TH01, TPOX, and vWA) were obtained using Precision ID GlobalFiler NGS STR Panel v2 from 82 unrelated individuals sampled from the Japanese population. Autosomal STR alleles designated by NGS and conventional capillary electrophoresis were found to be concordant except at D2S441 allele 9.

An increasing number of studies on massively parallel sequencing of mitochondrial DNA (mtDNA) have been reporting identification of various types of noise or off-target sequences. Herein, we report that an off-target haplotype (sequence length 192 bp) observed in MiSeq data of mtDNA at nucleotide position 16,209-16,400 was likely caused by polymorphic nuclear mitochondrial DNA sequences (NumtS). Buccal DNA samples from Volunteers #001-004 and Control DNA 007 were amplified with our multiplex system of the B (15,998-16,172), C (16,209-16,400), and E (30-289) regions using 2000 copies of mtDNA. A sample index was added using a Nextera XT index kit, and MiSeq Reagent Nano Kit v2 was used in 2 × 251 cycles on a MiSeq FGx. FASTQ files were analyzed by CLC Genomics Workbench 21.0.3. The extracted SAM files were analyzed using our original Excel macro to sum up the read counts as the phased variant calls for each region. An off-target haplotype differing at 19 sites against the revised Cambridge reference sequence was observed in Volunteer #001 (4 in 10 MiSeq data), Volunteer #002 (2 in 9), and Control DNA 007 (6 in 9). In a BLAST search, the sequence of the off-target haplotype matched perfectly to three polymorphic NumtS (Poly_NumtS_430 [KM281528.1], HSA_NumtS_587 [HE613849.1], and nuclear fossil [S80333.1]) and BAC clone of chromosome 11 (AC107937.2). The sequence also matched perfectly to a Filipino mtDNA (KC993973.1) which was inferred as a chimeric sequence of mtDNA and the HSA_NumtS_587. The sequence of the off-target haplotype was not contained in the latest human reference genome sequence (GRCh38.p13). In a phylogenetic tree, the off-target haplotype was genetically distant from modern human mtDNA and not directly connected to them. In conclusion, observed off-target haplotype amplified by our multiplex system was likely caused by Poly_NumtS_430 or HSA_NumtS_587.

In forensic mitochondrial DNA (mtDNA) analysis, quantitative PCR (qPCR) is usually performed to obtain high-quality sequence data for subsequent Sanger or massively parallel sequencing. Unlike methods for nuclear DNA quantification using qPCR, a calibrator is necessary to obtain mtDNA concentrations (i.e., copies/µL). Herein, we developed and validated a mtDNA quantification method based on a SYBR Green assay by following MIQE [Bustin et al., Clin. Chem. 55 (2009) 611-22] and other guidelines. Primers were designed to amplify nucleotide positions 16,190-16,420 in hypervariable region 1 for qPCR using PowerUp SYBR Green and QuantStudio 5. The optimized conditions were 0.3 µM each primer and an annealing temperature of 60 °C under a 2-step cycling protocol. K562 DNA at 100 pg/µL was converted into a mtDNA concentration of 16,400 copies/µL using linearized plasmid DNA. This mtDNA calibrator was obtained by cloning the synthesized DNA fragments of mtDNA (positions 16,140-16,470) containing a 100-bp inversion. The linear dynamic range of the K562 standard curve was 10,000-0.1 pg/µL (r

Forensic human identification (HID) laboratories occasionally encounter non-specific peaks generated by non-human DNA. Casework samples for human short tandem repeat (STR) profiling may be contaminated by animal DNA because of the specific environment or situation from which they were obtained. Validation studies for HID kits have reported that non-specific peaks generated from some animals are observed near the human amelogenin peak. In this study, we first revealed that DNA sequences associated with the non-specific peaks generated from animal DNA differ from one animal family to the other. However, non-specific peaks cannot be analyzed using the remainder of polymerase chain reaction (PCR) products left over from conventional HID kits when human and animal DNA are mixed. To overcome this issue, we have developed a novel analysis method of using non-specific peaks generated from animal DNA in human STR profiling to identify the source of contaminating animal DNA at the family level. The method applied here is termed as blocking PCR, which involves selective animal DNA re-amplification by blocking nontarget human amelogenin DNA amplification using an oligonucleotide probe that specifically binds to human amelogenin using the remaining PCR product from the HID kit. Our data demonstrated that HID and family discrimination among animals that are often encountered in forensic contexts could be performed simultaneously. This study enabled recovery of more information from limited quantities of casework samples contaminated with animal DNA, which would be useful for forensic HID scientists.

Probabilistic genotyping software based on continuous models is effective for interpreting DNA profiles derived from DNA mixtures and small DNA samples. In this study, we updated our previously developed Kongoh software (to ver. 3.0.1) to interpret DNA profiles typed using the GlobalFiler™ PCR Amplification Kit. Recently, highly sensitive typing systems such as the GlobalFiler system have facilitated the detection of forward, double-back, and minus 2-nt stutters; therefore, we implemented statistical models for these stutters in Kongoh. In addition, we validated the new version of Kongoh using 2–4-person mixtures and DNA profiles with degradation in the GlobalFiler system. The likelihood ratios (LRs) for true contributors and non-contributors were well separated as the information increased (i.e., larger peak height and fewer contributors), and these LRs tended to neutrality as the information decreased. These trends were observed even in profiles with DNA degradation. The LR values were highly reproducible, and the accuracy of the calculation was also confirmed. Therefore, Kongoh ver. 3.0.1 is useful for interpreting DNA mixtures and degraded DNA samples in the GlobalFiler system.

As DNA typing systems have become increasingly sensitive in recent years, probability distribution models for back, forward, double-back, and minus 2-nt stutter ratios have been desired to be considered in DNA evidence interpretation using specific software programs. However, experimental investigations have been insufficient, especially for forward, double-back, and minus 2-nt stutters. In this study, we experimentally reevaluated the probability distribution models for each stutter ratio in the typing systems of GlobalFiler™ PCR Amplification Kit and 3500xL Genetic Analyzer from Thermo Fisher Scientific. In addition, to enhance the reliability of longest uninterrupted stretch (LUS) values and corrected allele numbers used in previously developed models for stutter ratios using sequence information (i.e., LUS model and multi-seq model), we propose the weighted average of LUS values and corrected allele numbers based on the number of observations in sequence-based population data. Back stutter ratios demonstrated a positive correlation with allele numbers (allele model) in eight loci, LUS values (LUS model) in eight loci, and corrected allele numbers (multi-seq model) in five loci. The forward stutter ratios (FSRs) of D22S1045 followed the LUS model. FSRs other than D22S1045 and double-back stutter ratios followed the LUS model by considering multiple loci together. Minus 2-nt stutter ratios observed in SE33 and D1S1656 did not increase with the increase in the allele numbers. The adopted models for each stutter ratio can be implemented in software programs for DNA evidence interpretation and enable a reliable interpretation of crime stain profiles in forensic caseworks.

In short tandem repeat (STR) analysis, length polymorphisms are detected by capillary electrophoresis (CE). At most STR loci, mobility shift due to sequence variation in the repeat region was thought not to affect the typing results. In our recent population studies of 1501 Japanese individuals, off-ladder calls were observed at the D12S391 locus using PowerPlex Fusion in nine samples for allele 22, one sample for allele 25, and one sample for allele 26. However, these samples were typed as ordinary alleles within the bins using GlobalFiler. In this study, next-generation sequencing analysis using MiSeq was performed for the D12S391 locus from the 11 off-ladder samples and 33 other samples, as well as the allelic ladders of PowerPlex Fusion and GlobalFiler. All off-ladder allele 22 in the nine samples had [AGAT]11[AGAC]11 as a repeat structure, while the corresponding allele was [AGAT]15[AGAC]6[AGAT] for the PowerPlex Fusion ladder, and [AGAT]13[AGAC]9 for the GlobalFiler ladder. Overall, as the number of [AGAT] in the repeat structure decreased at the D12S391 locus, the peak migrated more slowly using PowerPlex Fusion, the reverse strand of which was labeled, and it migrated more rapidly using GlobalFiler, the forward strand of which was labeled. The allelic ladders of both STR kits were reamplified with our small amplicon D12S391 primers and their mobility was also examined. In conclusion, off-ladder observations of allele 22 at the D12S391 locus using PowerPlex Fusion were mainly attributed to a relatively large difference of the repeat structure between its allelic ladder and off-ladder allele 22.

Phenomena called allele dropouts are often observed in crime stain profiles. Allele dropouts are generated because one of a pair of heterozygous alleles is underrepresented by stochastic influences and is indicated by a low peak detection threshold. Therefore, it is important that such risks are statistically evaluated. In recent years, attempts to interpret allele dropout probabilities by logistic regression using the information on peak heights have been reported. However, these previous studies are limited to the use of a human identification kit and fragment analyzer. In the present study, we calculated allele dropout probabilities by logistic regression using contemporary capillary electrophoresis instruments, 3500xL Genetic Analyzer and 3130xl Genetic Analyzer with various commercially available human identification kits such as AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. Furthermore, the differences in logistic curves between peak detection thresholds using analytical threshold (AT) and values recommended by the manufacturer were compared. The standard logistic curves for calculating allele dropout probabilities from the peak height of sister alleles were characterized. The present study confirmed that ATs were lower than the values recommended by the manufacturer in human identification kits; therefore, it is possible to reduce allele dropout probabilities and obtain more information using AT as the peak detection threshold.

Sibling assessment using the 15 autosomal short tandem repeat (STR) loci included in the Identifiler® kit can be difficult when comparing an unidentified party to an alleged sibling. Therefore, we investigated the likelihood ratio (LR) and the total number of shared alleles (TNSA) for sibship determination using the 21 autosomal STR loci included in the GlobalFiler™ kit. We computationally generated the genotypes of 10,000 sibling pairs and 10,000 unrelated pairs based on previously reported allele frequencies of the 15 Identifiler loci and the remaining 6 GlobalFiler loci. The LR and the TNSA were then calculated in each pair using the 15 and 21 loci. Next, these calculations were applied to 22 actual sibling pairs. LR values ⩾ 10,000 were observed in 48% of the sibling pairs using the 15 loci and in 80% of the sibling pairs using the 21 loci. The TNSA distribution between siblings and unrelated pairs was more divergent in GlobalFiler than in Identifiler. TNSA values ⩾ 20 were found only in true siblings in Identifiler, while TNSA values ⩾24 in GlobalFiler. In Identifiler, all pairs with TNSA ⩾ 24 had LR values ⩾ 10,000 and the same was true in GlobalFiler for TNSA ⩾29. Therefore, increasing the number of loci is very efficient for sibship determination. The LR is most reliable for determining sibship. However, TNSA values may be useful for the preliminary method of LR values because LR value demonstrated a significantly positive correlation with TNSA value in both Identifiler and GlobalFiler.

Allele frequencies for 22 autosomal short tandem repeat loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, and D22S1045) were obtained from 1501 unrelated individuals sampled from the Japanese population.

Body fluid identification and individual identification are an important series of tests in usual criminal investigations. Recent reports have demonstrated a new approach using DNA/RNA co-extraction methods in which RNA for body fluid identification and DNA for short tandem repeat (STR) typing are extracted simultaneously from the same sample. This study evaluated a standard co-extraction kit, the AllPrep® DNA/RNA Mini Kit, in order to demonstrate the availability of the co-extraction procedure for those real-time polymerase chain reaction-based body fluid identification methods that we have validated previously. We demonstrated that the use of the Allprep Kit, for which we adjusted the lysis temperature to 56°C to improve extraction efficiency, can simultaneously extract sufficient RNA and DNA for body fluid identification and STR analysis; however, a longer incubation at a high temperature slightly affected the Δ Ct value of each target gene and appeared to be not as effective for DNA extraction from old stains as from 1-day-old stains. This method is promising for future forensic investigations because the use of this kit can reduce sample consumption for body fluid identification and DNA typing.

An experimental DNA extraction kit (new kit) was recently developed to extract DNA from degraded skeletal remains without the need for powdering the samples. We compared the utility of the new kit with the conventional phenol/chloroform method using real-time quantitative PCR and multiplex STR analysis. The new kit yielded large amounts of DNA from a compact bone fragment compared with the conventional phenol/chloroform method. We were able to extract sufficient DNA for STR analysis from 75% (3 of 4) and 60% (3 of 5) of the un-powdered tooth and bone samples, respectively, using the new kit. We were able to perform mini-STR analysis of the remaining samples using DNA extracted with the new kit. Furthermore, we successfully performed mitochondrial DNA sequencing of every sample. The new kit simplifies the DNA extraction procedure as it does not require powdering samples. Decreasing the number of procedural steps in DNA extraction will be beneficial in controlling DNA contamination in laboratories. Our results suggest that the new kit may be used for the simple, simultaneous extraction of DNA from multiple samples.

Information regarding the ancestral and geographical origins of biological evidence samples may be useful for crime investigators as they narrow down the possible donors of the sample. A method for simultaneous analysis of seven biallelic markers (M130, M131, M57, M125, M175, M122 and M134) was developed for forensic application. M57, M125 and M131 are included to identify haplogroups inferred as having originated in the Japanese archipelago. Our method employs allele-specific PCR and fragment analysis using fluorescently labeled primers and capillary electrophoresis. This method can be used to assign a haplogroup from both of degraded male DNA samples and DNA samples containing a mixture of female and male DNA by designing PCR primers that generate small amplicons and are highly specific for targets on the Y chromosome. A total of 1346 samples from Japanese males collected from the four major islands and Okinawa island were classified into seven Y binary haplogroups i.e., C-M130, C-M131, D-M57, D-M125, O-M175, O-M122 and O-M134, and a "no-mutation detected" group and their frequencies were 0.0617, 0.0565, 0.1441, 0.182, 0.3418, 0.11, 0.0847 and 0.0193, respectively. Samples of "no-mutation detected" were further analyzed by direct sequencing for identification of the major haplogroup to which they belong. Along with the haplogroup data, we report haplotype data for the 16 Y-STR markers included in the AmpFlSTR ® Yfiler™ PCR amplification kit (Applied Biosystems). These data will be useful in the prediction of haplogroups based on Y-STR haplotypes.

A total of 3046 males of Chinese, Malay, Thai, Japanese, and Indian population affinity were previously typed for the Y STR loci DYS19, DYS385 (counted as two loci), DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4 using the AmpFlSTR Yfiler kit. These samples were assessed for population genetic parameters that impact forensic statistical calculations. All population samples were highly polymorphic for the 16 Y STR markers with the marker DYS385 being the most polymorphic, because it is comprised of two loci. Most (2677 out of a total of 2806 distinct haplotypes) of the 16 marker haplotypes observed in the sample populations were represented only once in the data set. Haplotype diversities were greater than 99.57% for the Chinese, Malay, Thai, Japanese, and Indian sample populations. For the Y STR markers, population substructure correction was considered when calculating the rarity of a Y STR profile. An F(ST) value, rather than a R(ST) value, is more appropriate under a forensic model. Because the F(ST) values are very small within the Asian populations, the estimate of the rarity of a haplotype comprised of 10-16 markers does not need substructure correction. However haplotypes with fewer markers may require F(ST) corrections when calculating the rarity of the profile.

Short tandem repeat studies are powerful tools for parentage analysis and for identification of missing persons, victims of murder, and victims of mass fatalities when reference samples are unavailable. The primer in the Identifiler® kit failed to amplify an allele at the D19S433 locus, producing a silent (“null”) allele. The causal mutation is a base change (G>A) 32 nucleotides downstream from the 3′ end of the AAGG repeats. The silent alleles are problematical in parentage analysis because when transmitted, they can cause a parent–child inconsistency that is unrelated to Mendelian genetics. The inconsistency is sometimes termed an “apparent opposite homozygosity” and it produces false evidence of nonparentage. Alternative primers were designed to amplify the D19S433 locus alleles and they detect the silent allele. Frequencies of the (no longer) silent allele were determined to be 0.0114 in 176 people from Shizuoka (Honshu) and 0.0128 in 156 people from Okinawa.

A total of 1079 Japanese males were typed for the following 16 Y chromosomal short tandem repeat (Y-STR) markers: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448 using an AmpFlSTR® Yfiler™ PCR Amplification kit (Applied Biosystems). A total of 950 haplotypes for the 16 Y-STR markers were detected and, of these, 886 haplotypes were unique. The most frequent haplotype was found in 22 Japanese males. The haplotype diversity was 0.9992, indicating a high potential for differentiating between male individuals. There were 10 haplotypes with no allele detected at the DYS448 marker. Thus, the presence of such atypical haplotypes should be noted, when DNA typing results obtained from degraded DNA samples and/or DNA mixture samples from more than one male individual are being interpreted.

Sequence variation in hypervariable regions HV1 and HV2 in the control region of mitochondrial DNA (mtDNA) was studied in 100 unrelated Japanese individuals living in Miyazaki Prefecture (in southern Japan) by PCR amplification and direct sequencing. PCR and sequencing primer sets designed for the C-stretch region at around position 16189 in HV1 and position 310 in HV2 were used. Sequence comparison revealed the existence of 90 different haplotypes in 120 variable positions, and of these 82, were unique, 6 were observed twice and 2 were observed three times. Genetic diversity was estimated at 0.999 and the probability of two randomly selected sequences matching (Random Match Probability, RMP) was 1.24%. The C-stretch region in HV1 was observed in 35% of individuals. The average number of nucleotide differences was 9.61 for HV1 and HV2. The majority of sequence variations were substitutions, particularly transitions from thymidine to cytosine (42.6%). Sequence heteroplasmy was not found in this study. Based on the observed polymorphic sites in HV1 and HV2, haplogroups D4 (D4a), M7a, M7b, N9a were the most commonly observed clusters. The resulting data were compared with some existing Japanese mtDNA databases. The present data contribute to expansion of the Japanese mtDNA database, particularly the database for Miyazaki Prefecture. These results show that an mtDNA database is very useful in forensic examination for identification of individuals.

Short tandem repeat (STR) typing is widely used in forensic investigation. When the same DNA sample is analyzed with different STR typing kits, a typing discrepancy is occasionally observed. In this study, we examined the cause of a typing discrepancy in a sample at D5S818 locus. This sample was designated as 10, 12 using Identifiler(®) , Identifiler(®) Plus, GlobalFiler(®) , PowerPlex(®) 16HS, and PowerPlex(®) 18D, but as 9.3, 12 using PowerPlex(®) Fusion. Sequencing results indicated that the shorter allele in the sample had a deletion (U31Tdel) at 31 nucleotides upstream of the repeat region (AGAT)10 . This deletion was located in the binding site of the published D5S818 forward primer in PowerPlex(®) 16 and was only 9 and 11 nucleotides downstream of our estimated 5' end position of D5S818 forward primer in GlobalFiler(®) and PowerPlex(®) 18D, respectively. We also examined the effect of primer length on the heterozygous peak balance in this sample.

The (E)-3-methyl-2-hexenoic acid (E3M2H), an axillary odor-related compound, is known to occur in Caucasians. The aims of this study were to clarify whether E3M2H contributes to axillary odor in Asians and to quantify and compare individual levels of E3M2H. Quantitative determination of E3M2H was performed by means of gas chromatography-mass spectrometry of sweat extracted from the axillary areas of T-shirts worn for 24 h by Japanese subjects. The amount of E3M2H was 15.9-34.6 nmol/ml in six of 30 subjects. Our method succeeded in quantitative analysis of E3M2H from axillary sweat collected individually; we also showed that E3M2H could be detected in Asians. This is the first report in which the amount of E3M2H in axillary sweat was quantified on an individual basis and compared to reveal individual differences. The results of this study indicate that E3M2H might contribute to axillary malodor in Asians as well as Caucasians.

In 1966, a male (17 years old) was clinically examined at the National Institutes of Health (NIH) and diagnosed with Idiopathic Progressive External Ophthalmoplegia (IPEO). A muscle biopsy showing ragged-red fibers implicated mitochondrial involvement. Since the sequence of human mitochondrial DNA (mtDNA) was not determined until 1981, no genetic confirmation of the disease was possible at that time. In 1999, clinical reexamination and sequencing the entire mtDNA of the patient and living maternal relatives (mother and brother) indicated a progressive mitochondrial myopathy and the presence of the 4977 base pair (bp) deletion (the common deletion) in the patient.

D-dimer, a specific fragment resulting from degradation of cross-linked fibrin, is an essential marker for the diagnosis of disseminated intravascular coagulation (DIC). Rapid assay for D-dimer using monoclonal antibody coated-latex particles might be useful for discriminating between postmortem and antemortem blood in bloodstains. We tried to detect D-dimer in nine postmortem blood samples by the rapid latex agglutination assay and to quantify them automatically using the latex photometric immunoassay system. The results showed that all samples were positive and that their amounts of D-dimer were 335-2,800 microg/ml (the normal blood level

A genotype of JC virus (JCV) DNA extracted from human urine can serve not only as a means of elucidating human migration patterns, but also of tracing the origin of unidentified cadavers. If a JCV genotype is determined using a small amount of urine or urine stain from a given person, it may be useful in identifying the geographic area in which that person was born. In the present study, we amplified a 610-bp intergenic region (IG region) of JCV DNA extracted from 200 μl of each of 82 urine samples collected in Japan. JCV DNA was detected in 45 of 82 urine samples (54.9%) and genotypes were also determined by restriction fragment length polymorphism analysis (MY genotype: 13; X (CY) genotype: 25; unclear: 7). Additionally, 16 urine stains which had been prepared 3 months earlier using 100 μl of JCV-positive urine were examined. JCV DNA was detected in 11 of 16 urine stains (68.8%), and in this case also, it was possible to determine the genotype by the phylogenetic analysis after DNA sequencing. The present study indicates that JCV genotyping can be successfully conducted using only 100 μl urine stain and even 3 months after the urine stain was made. Thus, JCV genotyping shows promise as a new analytical method for narrowing down the native place of unidentified persons or cadavers from urine stains left at the scene.

BCG vaccination using the multiple puncture device (the Heaf gun) has been used in Japan and in the United Kingdom. The appearance of the BCG scars therefore differs from that caused by the conventional intradermal BCG vaccination, which is used throughout the world. The aim of this study was to investigate the usefulness of examining BCG scars to identify the nationalities of unidentified cadavers. We investigated the BCG vaccination program not only in Japan, but also in other countries, along with the relation between the BCG scar and nationality. The results showed that the countries which domestically make and use the Heaf gun are Japan (where it is the only method that has been in use since 1968), the United Kingdom (where it has been used, in part, since 1982), and South Africa (where it has been used, in part, since 1972). In addition, for the past 10 years, the Japanese Heaf gun method has been partially applied in the Republic of Korea and in Brazil. The Heaf gun scar can be clearly distinguished from the intradermal scar, and is visible throughout a person's life when good technique is used to administer the vaccination. If the Heaf gun scar is found on the left upper arm of an unidentified Asian cadaver, it is sure to be that of a Japanese. The findings of this present study indicate that the Heaf gun scar can be examined to identify the nationalities of unidentified cadavers.

The transmission of a C16,291C/T heteroplasmy in the HV1 region of human mitochondrial DNA (mtDNA) was examined in buccal cells from 13 maternally-related individuals across three generations and in additional tissues (hair, blood, or finger nails) from three members of this family. The ratio of C:T at nucleotide position (np) 16,291 showed wide intra- and intergenerational variation as well as tissue variation within individuals. Our results demonstrate that one or two sequence differences between samples in the mtDNA does not warrant an exclusion. To avoid false exclusions especially when comparing mtDNA from hair samples, we recommend the analysis of as many samples as possible in order to minimize the possibility that the detection of a rare polymorphism in a single sample would be considered an exclusion when it is really a match. The observation that the transmission of a mtDNA heteroplasmy from one individual to her offspring is likely to differ among the first-generation offspring and between that generation and subsequent generations lends further credence to the bottleneck theory of inheritance of human mtDNA.

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.

This report describes a forensic case work where both the anthropological analyses and the DNA analysis contributed to establish a link between the fragmented bones and a victim. A total of 54 bone fragments were excavated from a crime scene. The gender and stature of the bone fragments were estimated anthropometrically from the radius discovered. Two out of 54 fragments were presumed to be the parts of swine scapula from their gross morphology, and were supposed to have been buried for a longer period than the human bones from their surface conditions. These findings were ensured by the histological examination and UV illumination in the compact bones. A nucleotide sequence of the mitochondrial DNA (mtDNA) control region in the radius was identical to that of a victim's sister. By this case work, the importance of combined activity in forensic biology was confirmed.

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.

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.

Analysis for short tandem repeat (STR) loci is widely performed in forensic laboratories for human identification that utilizes commercially available kits that employ fluorescently labeled primers and capillary electrophoresis. With only a few exceptions, the sequences of the primers included in a kit are not disclosed by the kit manufacturers. Therefore, we developed a simple method to determine the 5′ end of the binding site of the primers included in commercial kits. Our method requires only custom primers and human genome sequence data and routinely used equipment and consumables. One or two custom primers are added to the PCR reaction mixture containing kit primers and input human DNA prior to amplification, and PCR products are separated by capillary electrophoresis after amplification. With this method we can determine which primer of the pair is fluorescently labeled and the 5′ end of the binding site of primers based on the changes in an electropherogram that are caused by the addition of the custom primer(s), and the human genome sequence data. This method is also useful for the determination of the shortest possible lengths of labeled kit primers.

Sensitivity and specificity of commercially available ABO blood grouping monoclonal antibodies for medical use were examined for applying them to forensic blood typing. We have applied some monoclonal antibodies to hemagglutination test, enzyme-linked immunosorbent assay (ELISA) and absorption-elution test. Hemagglutination titer of these antibodies distributed ranging from ×4 to ×512. All antibodies examined could be used for detecting ABO blood type from secretor's saliva by ELISA method and using five anti-A antibodies blood group substances could be detected from non-secretor's saliva. Using anti-A and anti-B antibodies blood type could be detected from secretor's semen but using any antibody blood type could not be detected from non-secretor's semen. We could not get a correlation between hemagglutination titer and sensitivity on ELISA detected from saliva and semen. At absorption-elution test, using most of the anti-A and anti-B antibodies blood type could be detected of bloodstains and secretor's body fluid stains. Using several antibodies blood type could not be detected of non-secretor's samples. Using all of anti-A antibodies and some of anti-B antibodies it was failed to detect blood type of hair samples. Using all of the anti-H antibodies blood group substances could not be detected of stain or hair samples. This result suggested that we have to select suitable monoclonal antibodies of ABO blood grouping for each evidential sample and each blood typing method.