Kentaro Kasai

Amplification of a Variable Number of Tandem Repeats (VNTR) Locus (pMCT118) by the Polymerase Chain Reaction (PCR) and Its Application to Forensic Science

A genetic locus (D1S58, defined by DNA probe pMCT118) that contains a variable number of tandem repeats (VNTR) has been successfully amplified from a very small amount of genomic deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR). The DNA sequence of the locus was determined and was found to consist of a 16-base consensus sequence and flanking sequences. Oligonucleotide primers complementary to the flanking sequences were synthesized to serve as primers for amplification of MCT118 by the PCR method. Human genomic DNA isolated from blood (2 ng from each sample) was successfully amplified at the MCT118 locus, and polymorphic bands were detectable by ethidium bromide staining after electrophoresis on polyacrylamide gels. Determination of genotypes at this VNTR locus can now be routinely achieved within 24 h, without the need for Southern blots or radioactive materials. Furthermore, the small size (387 to 723 base pairs) of the DNA fragments produced in the PCR amplification permits good resolution of individual alleles that differ by only one repeat unit. The precise specification of the number of tandem repeats present in each allelic fragment is reproducible from one analysis to another.

The modified method of two-step differential extraction of sperm and vaginal epithelial cell DNA from vaginal fluid mixed with semen

This investigation was undertaken as an efficient method for isolating sperm DNA from a mixed fluid sample which contains vaginal epithelial cells in a greater amount. The modified method of the two-step differential extraction procedure was found to be suitable for separating sperm DNA and vaginal epithelial cell DNA from the mixed stains. As the first step of digestion, vaginal epithelial cells in the mixed stains were lysed with Proteinase K and SDS, and sperm heads remaining in the lysed solution were collected by centrifugation. As the second step digestion, the sperm heads were lysed with the buffer containing Proteinase K, SDS and DTT as reducing agent. DNA fractions extracted from the two lysed solutions were enriched, one with sperm DNA and the other with vaginal epithelial cell DNA. MCT118(D1S80), ApoB VNTR and HLADQ alpha types of sperm DNA were detected and were confirmed by matching with corresponding male blood DNA. In the case of vaginal secretion mixed with semen of two males, the mixture of MCT118 types of the two males was detected in sperm DNA fraction.

Inter- and intragenerational transmission of a human mitochondrial DNA heteroplasmy among 13 maternally-related individuals and differences between and within tissues in two family members ☆

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.

Role of p38 mitogen-activated protein kinase on cardiac dysfunction after hemorrhagic shock in rats.

Cardiac dysfunction is a well-known complication of hemorrhagic shock as a consequence of local inflammatory response. Several studies have indicated that p38 mitogen-activated protein kinase (MAPK) is a key mediator in organ dysfunction that is associated with the inflammatory state through the activation of proinflammatory cytokines such as TNF-&agr; and IL-1&bgr;. Whether the same applies to cardiac dysfunction after hemorrhagic shock has not been clearly determined. Therefore, in this study, the role of p38 MAPK on cardiac dysfunction after hemorrhagic shock was studied up to 5 h after a hemorrhage using FR167653, a specific inhibitor of p38 MAPK phosphorylation. The p38 MAPK phosphorylation, the cardiac mRNA expressions of TNF-&agr; and IL-1&bgr;, and intracardiac serum concentrations of each cytokine and creatine phosphokinase-MB isozyme increased after a hemorrhage. Activated neutrophil accumulation in the heart, histological inflammation-related injuries, and frequent ventricular arrhythmia were observed in the late phase after hemorrhagic shock. FR167653 inhibited these hemorrhagic changes except the induction of the primary hypotensive state. These results demonstrate that p38 MAPK phosphorylation in hemorrhagic shock plays an important role in the cardiac expression of the proinflammatory cytokines and in the development of cardiac dysfunction relative to the inflammatory responses.

Evaluation of new primers for CSF1PO

We describe new primers for the detection of the STR polymorphism at the CSF1PO locus. These primers have been designed to produce shorter amplicons (150–182 bp) than the primers in standard use (295–327 bp). The reliability of the new primers for CSF1PO typing has been demonstrated by testing on known samples and by sequence analysis. These primers are superior to the original primers with regard to electrophoretic resolution and utility for typing of severely degraded DNA.

Mitochondrial DNA heteroplasmy among hairs from single individuals.

A denaturing gradient gel electrophoresis (DGGE) assay was used to detect mitochondrial DNA (mtDNA) sequence heteroplasmy in 160 hairs from each of three individuals. The HV1 and HV2 heteroplasmic positions were then identified by sequencing. In several hairs, the heteroplasmic position was not evident by sequencing and dHPLC separation of the homoduplex/heteroduplex species was carried out with subsequent reamplification and sequencing to identify the site. The overall detection frequency of sequence heteroplasmy in these hairs was 5.8% (28/480) with DGGE and 4.4% (21/280) with sequencing. Sequence heteroplasmy of hair was observed even when the reference blood sample of the individual was homoplasmic. The heteroplasmic positions were not necessarily observed at sites where high rates of substitution have been reported. In two hairs, a complete single base change from the reference blood sample was observed with sequencing, while the heteroplasmic condition at that site in the hair was observed using DGGE. The DGGE results in such samples would serve as an aid in considering the possibility of match significance. In a forensic case, this situation would lead to the possibility of a failure to exclude rather than to be inconclusive.

A D19S433 Primer Binding Site Mutation and the Frequency in Japanese of the Silent Allele It Causes

Abstract:  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.

The effects of Asian population substructure on Y STR forensic analyses

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.

Role of p38 mitogen-activated protein kinase pathway on renal failure in the infant rat after burn injury.

The p38 mitogen-activated protein kinase (MAPK) pathway is a proinflammatory signal transduction pathway for the production of cytokines and cellular response to stress, such as bacterial LPS or ischemia. We examined the effects of FR167653, a specific inhibitor of p38 MAPK, to explore the relationship between intestinal barrier damage and remote renal dysfunction. Immunohistochemical data showed the accumulation of neutrophils in the intestine after burn, and a horseradish peroxidase (HRP) tracer experiment showed burn-induced intestinal barrier damage. Our quantitative bacterial culture data demonstrated that viable bacteria reached the remote organs after burn and prevented the invading viable bacteria from using FR167653. Western blotting identified increased phosphorylation of p38 MAPK in the kidney after burn, and it may also have shown the possibility that endotoxin associated with the bacterial translocation enhances the activation of the p38 MAPK pathway. We blocked the intestinal barrier damage using FR167653, which resulted in reduced neutrophils in the intestine. FR167653 also prevented the increased phosphorylation of p38 MAPK in the kidney, which resulted in reduced neutrophils in the glomerulus and the reduction of tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA in the kidneys, and, finally, prevented burn-induced renal failure. This study provides evidence for the hypothesis that the p38 MAPK pathway controls inflammatory mediators and not only improves intestinal function but also reduces remote renal failure after burn. We identified the pathophysiologic role of the p38 MAPK pathway in the development of renal failure after burn.

Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a non-powder method.

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.