Hirofumi Tsutsumi

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.

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.

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.

Real-time PCR assay for the detection of picoplankton DNA distribution in the tissues of drowned rabbits

The detection of plankton DNA is one of the important methods for the diagnosis of drowning from postmortem tissues. This study investigated the quantities of picoplankton (Cyanobacteria) DNA in the lung, liver, kidney tissues and blood of drowned and non-drowned rabbits, and the sensitivity of detection of picoplankton DNA by polymerase chain reaction (PCR) detect for the diagnosis of death from drowning. For this purpose, the DNA of the 16S ribosomal RNA gene of picoplankton was quantitatively assayed from the tissues of drowned and non-drowned rabbits immersed in water after death. Each of the liver, kidney and lung tissues and blood were obtained from drowned and non-drowned rabbits. Picoplankton DNA in the tissues was extracted using the DNeasy® Blood & Tissue kit to determine the yield of picoplankton DNA from each tissue. TaqMan real-time PCR was performed for quantitative analysis of picoplankton DNA. Target DNA was detected in the liver, kidney and lung samples obtained from the drowned rabbits, while no picoplankton DNA was detected in the non-drowned rabbit tissues (except in lung samples). The results verified that direct PCR for the detection of picoplankton DNA is useful for the diagnosis of drowning. Although we observed seasonal changes in the quantity of picoplankton in river water, we were able to detect DNA from various organs of drowned bodies during the season when picoplankton were not the most abundant.

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.

Sex Determination with a Discriminate Function Analysis of Deciduous Teeth Size in Plaster Models

Plaster models of the teeth of 3-year-old Japanese children (96 males, 98 females) were used to record the crown length, crown width and crown thickness of 5 maxillary and 5 mandibular deciduous teeth (30 measurement values). These measurements were used to devise a number of sex determination formulae. A sex-determination formula using all 30 values was calculated. Furthermore, a number of practical formulae were derived from only the crown width and crown thickness values because the deciduous teeth wear in 4 years and older children rapidly progress, making the crown length measurement unreliable. These formulae were calculated for the maxillary teeth alone and mandibular teeth alone. The formulae based on only the crown width or thickness were also calculated for both maxillary and the mandibular teeth. A step-wise discriminant analysis was then used to ascertain the most reliable measurements and a practical formula subsequently devised. The results obtained were as follows: 1. The mean value for each measurement was greater in males than in females. 2. Significant differences in the values recorded were seen in 28 out of the 30 measurements taken. The measurement items not exhibiting these significant differences were the crown width of the maxillary lateral incisor and the crown thickness of the mandibular second molar. 3. The accuracy rates for the sex-determination analysis and the step-wise sex determination analysis calculated using all 30 values were 78.6% and 75.7%, respectively. 4. The accuracy ranges for the modified sex-determination formulae and the associated step-wise sex determination analyses were 70.6-78.4% and 67.0-76.8%, respectively.