Yusuke Doi

Typing of Y chromosome single nucleotide polymorphisms in a Japanese population by a multiplexed single nucleotide primer extension reaction

We have developed a new method for typing single nucleotide polymorphisms (SNPs) on the human Y chromosome based on a multiplexed single nucleotide primer extension. This method has the advantage that several SNPs are typed rapidly and simultaneously. We examined 15 different SNP loci on Y chromosome, M9, M105, M122, M125, M128, M130, SRY465, IMS-JST006241, IMS-JST006841, IMS-JST002611, IMS-JST003305, IMS-JST008425, IMS-JST021354, IMS-JST021355 and IMS-JST055457, in 159 Japanese males. From the typing results of these 15 loci, we found 13 haplotypes. Gene diversity for each locus ranged from 0.025 to 0.486 and the haplotype diversity was estimated to be 0.838. This method could be readily applied for personal identification and paternity testing.

Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay

Saliva is one of the most common body fluids found at a crime scene. Therefore, identifying saliva is important in forensic science. However, the current protein marker assays used to identify saliva are not sufficiently specific. Although proline-rich proteins (PRPs) are highly specific for saliva, their forensic potential has not yet been investigated. In this study, we developed enzyme-linked immunosorbent assays (ELISAs) to detect acidic salivary PRP HaeIII subfamily 1/2 (PRH1/2) and basic salivary PRP 2 (PRB2). The specificity, sensitivity, and efficiency of the ELISAs for PRH1/2 and PRB2 were compared with those of the ELISA for statherin (STATH), a known protein marker for saliva. The levels of PRH1/2 were significantly higher in saliva and saliva stains than in other body fluids (nasal secretions, urine, semen, vaginal fluid, blood, and sweat). PRB2 and STATH were detected in both nasal secretions and saliva. The PRH1/2 ELISA showed sensitivity similar to that of STATH ELISA. The detection rate of PRH1/2 ELISA was almost similar to that of STATH ELISA, followed by the ELISA for PRB2. The PRH1/2 ELISA had higher specificity for saliva than STATH ELISA. Therefore, the PRH1/2 ELISA has potential as a method to identify saliva for forensic investigation.

Polymorphism of the D12S391 microsatellite in a Japanese population sample

Using the polymerase chain reaction (PCR), we studied the short tandem repeat (STR) polymorphism observed at the D12S391 locus. In 350 Japanese examined, 14 different alleles ranging from 209 bp to 261 bp were detected. Allele 18 (221 bp) showed the highest frequency at 0.30. Observed and expected values of respective genotypes satisfied the Hardy-Weinberg equilibrium (chi 2 = 24.08, P = 0.24, df = 20). In addition, 18 additional sequence structures (suballeles), were detected in this study. Within the suballeles, sequence variants, in which the initial repeat of (AGAT) was replaced with (AGGT), was found in five samples. It was found that the analysis of single-strand conformation polymorphism (SSCP) before sequence analysis was useful for distinguishing these suballeles.

Detection of fatty acid-binding protein 5 and small proline-rich protein 3 for forensic vaginal fluid identification by ELISA

Vaginal fluid identification is often required for forensic investigation of sexual assault cases. However, standardized assays for vaginal fluid identification have not been developed. Recently, we identified human fatty acid-binding protein 5 (FABP5) and human small proline-rich protein 3 (SPRR3) as characteristic vaginal fluid proteins by performing peptide mass fingerprinting. In this study, we developed enzyme-linked immunosorbent assays (ELISAs) for detecting FABP5 and SPRR3 and evaluated the specificity and sensitivity of these assays for detecting vaginal fluid. The data indicate that the levels of both protein markers were significantly higher in vaginal fluids and vaginal fluid stains than in other body fluids (nasal secretions, saliva, urine, semen, blood, and sweat). The dilution limits of FABP5 and SPRR3 ELISAs equated to 0.06 μL and 0.03 μL, respectively, of vaginal fluid extracts, thought to be sufficient for application to real forensic samples. Furthermore, the levels of both protein markers are not lowered during the menstrual cycle. The protein markers were also detectable in menopausal samples, taken from menopausal and pregnant volunteers. The protein markers were detected in some aged stains. FABP5 ELISA showed a better detection rate in inter laboratory tests using simulated casework samples compared to SPRR3 ELISA. Overall, FABP5 can be more useful for the identification of vaginal fluid for forensic investigation, although both FABP5 and SPRR3 assays can potentially be useful.