An improved method for the diatom test utilizing DNA binding ability of silica.

In order to devise a better forensic test for diatoms, the DNA binding ability of the diatom frustule constructing by silica, in the presence of chaotropic ions were utilized. It was proved that the diatoms were able to be captured via λDNA using silica-coated magnetic beads (Mag beads), followed by isolation and purification from the Mag beads as a solid phase by substituting the chaotropic agent with ultrapure water. Five cases of drowning, three in freshwater and two in seawater, were applied to the present method and similar results as the usual diatom test were obtained. Specimens of lung and other organs were rendered clearly visible, with elimination of foreign impurities. The present method appears applicable for detection of diatoms indirectly using PCR amplification of bound DNA or directly staining of the DNA.

A simple DNA coprecipitation method for the detection of diatoms in heart blood.

We developed a method for detecting and enumerating diatoms in the heart blood of drowning victims and evaluate its utility for diagnosing death by drowning. For purification of diatoms from blood, the DNA binding ability of the diatom frustule in the presence of a chaotropic agent was utilized. The procedure is basically the same as the commonly used method for DNA purification from blood using Proteinase K treatment and denaturation by a chaotropic agent. DNA adsorbed to the diatom (DNA/diatom complex) is recovered by ethanol precipitation, and the DNA is subsequently digested using DNase. Purified diatoms could be clearly observed under a microscope. Diatoms spiked in the blood of non-drowned cadavers (n=15) were well recovered, and were detected in heart blood from all drowning victims (n=22). The mean number of diatoms found in 5 ml of blood from drowning victims was 7.8±5.8 (mean±SD), and the number of diatoms detected in the blood of the left ventricle (6.1±5.8) was approximately two times higher than that of the right ventricle (3.0±2.7, p<0.05). These results suggest that this simple and safe method can become an effective tool for diagnosing the cause of death as drowning.

STR and mitochondrial DNA SNP typing of a bone marrow transplant recipient after death in a fire.

Personal identification of a house fire victim is described. About 5 years prior to death, the victim had been underwent bone marrow transplantation (BMT) with a graft from an unrelated donor as treatment for acute myelogenous leukemia. Clinically, the victim had been in remission at the time of death. Typing of STRs and sequencing of mitochondrial DNA (mtDNA) were performed using blood from the heart as well as several soft (psoas major muscle, uterine muscle and mucous membrane of the urinary bladder) and hard (costal cartilage and nail) tissues. STR genotypes and amelogenin from each of the tissue samples were successfully typed, and the parentage was identified. The blood STR types demonstrated no relationship with those from other tissues. None of the blood STR loci showed extra peaks arising from those of the recipient. Therefore, the blood stem cells were assumed to have been altered to those of the donor. The genotypes of mtDNA control regions were also examined. The electropherogram of hypervariable region II (nucleotide positions 29-408) obtained from the blood revealed a similar length heteroplasmy, suggesting microchimerism of the blood. Sequence analysis of mtDNA might be applicable as a more sensitive method for determination of chimerisms after BMT.

Marine bacteria comprise a possible indicator of drowning in seawater.

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples (n=20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater (n=8) and in fresh water (n=7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n=7). Bioluminescent colonies were detectable on 2-4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria (Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.

Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region.

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.

Establishment and characterization of a novel human pancreatic cancer cell line (SUIT-4) metastasizing to lymph nodes and lungs in nude mice.

A new tumor cell line (SUIT-4) derived from ascites of a patient with carcinoma of the pancreas has been established in tissue culture and in nude mice, and maintained for over 7 years. In tissue culture, the cells grew as a confluent monolayer with piling up of cells in some areas. The population doubling time during the exponential phase of the cell growth was 43.9 h in vitro. Chromosome count ranged from 63 to 68 with a modal number of 67. Subcutaneous injection of cultured cells into the flanks of nude mice resulted in tumor formation with a doubling time of 88.8 h. Histopathologically, xenografts in nude mice were moderately differentiated tubular adenocarcinoma, and the tumor cells showed spontaneous metastasis to the regional lymph nodes in 6 of 21 nude mice and to the lung in 4 of 21. Transmission electron microphotographs confirmed the ductal cell origin of the carcinoma and revealed that the cells had abundant mitochondria and lysosomes. SUIT-4 cells released carcinoembryonic antigen (3.08 x 10(2) ng/1 x 10(6) cells/24 h) and carbohydrate antigen 19-9 (4.75 x 10(4) U/1 x 10(6) cells/24 h) during exponential cell growth in vitro. Reverse transcriptase-polymerase chain reaction studies revealed that SUIT-4 cells expressed matrix metalloproteinases 1, 3, 7, 10 and 14.

A method for genotyping Y chromosome-linked DYS385a and DYS385b loci.

We developed a method for genotyping Y chromosome-linked homologous DYS385 loci individually, combining locus-specific polymerase chain reaction (PCR) amplification and previously reported procedures. Duplicated DYS385a (5'-end) and DYS385b (3'-end) loci were located about 41 kb apart and inverted to each other in the Y chromosome, which data was obtained from the human genome sequence in National Center for Biotechnology Information (NCBI), and sequence differences were found at 424 bp down- and upstream of each locus. The locus-specific amplifications were performed using primers designed for this intergenic region, and fragments about 900 bp in length were produced. Polymorphic tetranucleotide arrays in the PCR products were typed according to procedures reported previously. Twenty male subjects were genotyped using this method. Alleles (GAAA)13-(GAAA)21 were observed at the DYS385a locus, and those at DYS385b included alleles (GAAA)9-(GAAA)15. DYS385a alleles, excluding those of identical arrays, were always larger than DYS385b alleles in the same subjects. These data suggest that the DYS385a and DYS385b loci can be amplified completely for discrimination, and the genotypes of the alleles provide information useful for forensic case work and population genetics.

Mitochondrial DNA and STR typing of matter adhering to an earphone.

STR typing and mitochondrial DNA (mtDNA) sequencing were performed on the matter adhering to an earphone found at a crime scene. Experimental studies were carried out using the earphones provided by volunteers. By means of immunohistochemistry, keratinocytes and a portion of nucleated epithelial cells were proven to exist in the contents from the earphones. DNA was extracted by means of the phenol/chloroform method, and the low quantity of extracted DNA was found to be highly degraded. Six STR loci, CSFIPO, TPOX, TH01, F13A01, FESFPS and vWA, were PCR amplified and typed by using two triplex systems (CTT and FFv Multiplexes, Promega, WI), and an amelogenin locus was determined as well. Although partial profiles were observed in some experimental samples, all STR loci could be typed when a considerable amount of high molecular weight DNA was obtained (>0.5 ng/microL). Amplification and sequencing of mtDNA hypervariable region I(15997-16401) and hypervariable region 11(29-408) were all successful. The mitochondrial DNA sequence of the actual case sample, comprising two hypervariable regions and a total of 785 base pairs, showed eight mutations and two insertions with respect to the standard published reference sequence. The genotype was unique in the three published Japanese databases. These results suggest that it is possible to analyze mtDNA from minute amounts of materials and from degraded materials more effectively and routinely in forensic practice.