Tomohiro Takayama

Determination of deleted regions from Yp11.2 of an amelogenin negative male

The use of short tandem repeat (STR) multiplexes with the incorporated gender marker amelogenin is now common practice in forensic laboratories. The amelogenin locus is encoded by two single copy genes located on Xp22.1-Xp22.3 (AMELX) and Yp11.2 (AMELY). There are differences in size and sequence between AMELX and AMELY that can be used for sex-typing tests. A sized-based difference for AMELX and AMELY is an integral part of most PCR multiplex kits that are used for DNA profiling. However, we experienced a case of a normal male being typed as female (dropout of the amelogenin Y allele) with AmpFlSTR Profiler kit, AmpFlSTR Identifiler kit and PowerPlex 16 System. Further testing with Y-STR loci using the AmpFlSTR Yfiler kit revealed an additional null at DYS458 locus in this amelogenin negative male. We examined the deleted regions using a total of 60 loci from Y-STRs, STSs (sequence tagged sites) and newly designed primer sets. Three deleted regions in Yp11.2 were seen in this sample. The sizes of these deletions were approximately 2.51 Mb, 25 kb and 834 b, respectively. The deletions did not belong to the five reported patterns in a collection of 45 deletion males from 12 populations described by Jobling et al.

DNA typing from cigarette butts

We performed DNA typing for D1S80, HLADQA1, TH01 and PM using the butts of 100 cigarettes that were smoked by ten different individuals (ten cigarettes per individual). The results obtained from DNA typing for D1S80 agreed with the results obtained using bloodstains in 76 cigarette butt samples. Sixteen samples produced false results, showing the loss of the longer allelic hetero-band. When examined using agarose gel electrophoresis, high-molecular weight DNA was not observed in these samples. The same results were also observed for buccal swab samples and saliva stains obtained from the same individuals. In the remaining eight cigarette butt samples, PCR products were not detected. The results obtained from DNA typing for TH01, HLADQA1 and PM agreed with the results obtained using bloodstains in 90 samples. In the remaining ten samples of a specific kind of cigarette (Marlboro), the PCR products were not detected. The extracts from the ends of the Marlboro cigarettes were stained yellow. When the DNA extracted from Marlboro cigarette butts was treated with Microcon-100 (amicon) or SizeSep 400 Span Columns (Amersham Pharmacia Biotech), PCR products could be detected. When PCR amplification was performed after adding extracts from the ends of unsmoked Marlboro cigarettes to DNA extracted from bloodstains, PCR products could not be detected. The present data indicate that the degradation of high-molecular weight DNA and the inhibition of PCR by dyes of the cigarette end should be kept in mind when performing DNA typing using cigarette ends.

Japanese Population DNA Typing Data for the Loci LDLR, GYPA, HBGG, D7S8, and GC

Population studies on the five loci LDLR, GYPA, HBGG, D7S8, and GC (PM loci) were carried out in a sample of 366 unrelated Japanese individuals living in Gifu Prefecture (central region of Japan) using the AmpliType PM PCR Amplification and Typing kit (Perkin Elmer). For all loci, no significant deviations from Hardy-Weinberg equilibrium could be found in our Japanese population sample. The combined mean exclusion chance and power of discrimination for the PM loci were 0.68 and 0.993, respectively. The Japanese and Chinese population data were similar for the all loci. The Japanese and Korean population data were similar for all loci other than D7S8. Significant differences were observed between the Japanese population data and the 16 other population data compared for 3 loci or more.

Origin of DNA in human serum and usefulness of serum as a material for DNA typing.

The aims of this study were to clarify the origin of DNA in human serum and to investigate whether serum is a material available for DNA typing in routine forensic practice. Blood was donated from 10 healthy adult volunteers and stored for up to 8 days, at 4 degrees C and at room temperature. The serum DNA concentration at zero time was in the range of 5.6 to 21.8 ng/ml with a mean of 12.2+/-1.6 ng/ml. The concentrations increased with storage time. On agarose gel electrophoresis, all serum samples showed ladder patterns and the size of each band was an integer multiple of approximately 180 bp considered to be characteristic of apoptosis. DNA typing from DNA released by apoptosis was possible. Exact DNA typing of D1S80, HLA DQA1, PM, CSF1PO, TPOX, TH01 and vWA was possible for each sample. These results indicate that serum contains fragmented DNA derived from apoptosis of leukocytes, especially neutrophils, and that fragmented DNA is an appropriate material for DNA typing.

Development of an indirect competitive ELISA for the detection of ABO blood group antigens

We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of ABO blood group antigens in human samples; in particular for blood stains. ABO blood group antigens conjugated to polyacrylamide were used for immobilized antigen. ABO blood group antigens were extracted from blood stains using a novel method involving pre-incubation with proteinase K (PK), followed by heat treatment. The extracts (analytes) were combined with either anti-A or -B monoclonal antibodies (mAbs), and added directly to the antigen-coated wells. The anti-A and -B mAbs were captured by either ABO blood group antigens present in the analyte or by the immobilized blood group antigens. Peroxidase-conjugated anti-mouse IgM antibody was used to detect anti-A and -B mAbs complexed with immobilized blood group antigens, and a colorimetric reaction using o-phenylenediamine/H2O2 used for its measurement. The ELISA developed in this study was able to detect blood group antigens in blood, saliva and blood stains. The detection limit for unknown blood, saliva and blood stain were determined as 1:200, 1:32 and 1:16. Overall the ABO blood grouping ELISA can be used with relative ease for the high throughput screening of biological samples for the detection of ABO blood group antigens.

The STR locus D11S554: allele frequencies and sequence data in a Japanese population

Abstract A population study on the short tandem repeat (STR) locus D11S554 was carried out in a sample of 362 unrelated Japanese individuals living in the Gifu Prefecture. A total of 46 different alleles ranging from 180 bp to 340 bp and 135 genotypes were revealed. Sequence analysis of alleles was carried out for 185 samples. The sequence structures of the repeat regions of the alleles were found to be complex and the alleles were classified into nine sequence types, including four new sequence types. According to the system of Adams et al. (1993), we designated the new sequence types IA3, IA4, IA5 and IB3, respectively. Out of the 46 different alleles, 11 showed sequence heterogeneity. The results of this study demonstrated that the D11S554 locus is a powerful and useful genetic marker for forensic practice in the Japanese population.

Blood grouping of mixed bloodstains using immunocytochemical methods.

Immunocytochemical methods to determine the ABO blood group of each blood of mixed bloodstains have been developed. Mixed bloodstains were made on surgical blades and a cedar board. The blades were dipped into blood and then dipped into blood of a different group at intervals of 30, 20, 15, 10 and 5 s. Two drops of blood were dropped on a cedar board and then two drops of blood of a different group were dropped there at the same intervals. The bloodstains were dried for a week. The blood samples were removed from the blades or the cedar board and processed according with a routine histological method. Three serial thin sections were obtained. After deparaffinization, the sections were treated in papain solution for 2 h at 36 degrees C, to unmask antigenic sites on red cell membranes. The labeled streptavidin-biotin (LSAB) and peroxidase-anti-peroxidase (PAP) methods were used to detect A and B antigens, and an indirect immunocytochemical method for H antigen. These immunocytochemical methods showed specific immunologic reactions and allowed determination of the blood group of each blood of mixed bloodstains. Further, these methods indicated a possibility to determine who was stabbed first, in cases where two or more victims were stabbed with a single knife.

Involvement of soil bacteria in ABO blood mistyping

The current study investigated whether ABO blood mistyping of human biological samples is induced by soil bacteria. A total of 380 bacterial strains were isolated from 50 discrete soil samples using human blood agar, and glycosidase activity evaluated for all strains using 4-nitropheny glycosides (4-nitrophenyl n-acetyl-α-D-galactosaminide, 4-nitrophenyl-α-D-galactopyranoside, 4-nitrophenyl-α-L-fucopyranoside) as substrates. Thirteen strains possessed α-galactosidase activity, and 16S rRNA sequence analysis revealed a close relatedness to the genus Bacillus. An indirect competitive enzyme-linked immunosorbent assay confirmed seven strains exhibited type B antigen degradation activity. These results demonstrated that 1.8% of the bacteria isolated from soil, were Bacillus sp., possessed galactosidase activity, and had the potential to cause ABO blood mistyping.