Jian Tie

Genetic analysis of twelve X-chromosomal STRs in Japanese and Chinese populations

We investigated the genetic markers of twelve X-STR loci in 670 healthy, unrelated Japanese (438 men and 232 women) from Tokyo and 488 Chinese (263 men and 225 women) from Shenyang, using the Investigator Argus X-12 kit. Allele and haplotype analyses of twelve X-STRs clustered into four linkage groups indicated that they are highly informative for forensic applications in Japanese and Chinese populations. Hardy–Weinberg equilibrium tests demonstrated no significant deviations in the two populations. Among the four closely linked X-STR trios, some haplotype unique to Japanese or Chinese population were detected. Haplotype diversity for each linkage group ranged from 0.9861 to 0.9968, showing high values in each of the study populations. The genetic distances between populations based on the 12 X-STR loci and the phylogenetic tree revealed long genetic distances between Asian and Caucasian populations and between Asian and African population (Moroccan). These results suggest that the twelve X-STR loci will contribute to forensic casework in Japanese and Chinese populations.

Genetic polymorphisms of eight X-chromosomal STR loci in the population of Japanese

The genetic polymorphisms of eight X-chromosomal short tandem repeats (STR) DXS10135, DXS8378, DXS7132, DXS10074, HPRTB, DXS10101, DXS10134 and DXS7423 were analyzed in a sample of 492 unrelated males (283) and females (209) from the Japanese population. Multiplex PCR amplification was performed using the Mentype Argus X-8 PCR amplification kit. The haplotype frequencies within the four linkage groups were studied for the 283 examined Japanese males. Allele frequencies of eight X-STR loci were calculated separately for males and females, and exact tests demonstrated no significant deviations from Hardy-Weinberg equilibrium. Several microvariant and rare alleles were observed, and forensic efficiency parameters were calculated. The combined powers of discrimination of the loci in men and women were 0.999995 and 0.9999999999988, respectively.

Direct and rapid PCR amplification using digested tissues for the diagnosis of drowning

We developed a direct and rapid method for the diagnosis of death by drowning by PCR amplification of phytoplankton DNA using human tissues. The primers were designed based on the DNA sequence of the 16S ribosomal RNA gene (16S rDNA) of Cyanobacterium. Samples of lung, liver and kidney tissues were collected from 53 autopsied individuals diagnosed as death by drowning. Without DNA extraction, the tissue fragments were incubated directly in a digest buffer developed in this study, for 20 min. Using 1 μL of the tissue digest solution in PCR, the 16S rDNA was successfully amplified. The specific 16S rDNA fragment was identified from the standard picoplankton Euglena gracilis, the tissues of bodies died from drowning and water samples from the drowning scenes. On the other hand, no PCR products were found in the tissues of individuals who died from causes other than drowning. Various quantities of tissue weighing 1, 5, 10, 20 and 30 mg were tested, and the PCR amplification detected the specific16S rDNA fragment from all the quantities of tissue tested. This method was found to be more reliable, sensitive, specific and rapid when compared to the conventional diagnosis of death by drowning using the diatom test by acid digestion method.

Detection of short tandem repeat polymorphisms from human nails using direct polymerase chain reaction method

Human nail is an important forensic material for parental testing and individual identification in large‐scale disasters. Detection of STR polymorphism from hard tissues generally requires DNA purification, which is technically complicated and time consuming. In the present study, we attempted to detect STR polymorphisms from untreated human nail samples by direct PCR amplification method using the primer mixture supplied with the GenePrint® SilverSTR® III System or the AmpFℓSTR® Identifiler® PCR Amplification Kit, and Tks Gflex DNA polymerase known to be effective for amplification from crude samples. A nail fragment measuring approximately 1.5 mm in breadth and 0.5 mm in length was placed directly into a PCR tube, and various PCR conditions were tested. The PCR products were analyzed by denaturing acrylamide gel electrophoresis or CE. Multiple STR polymorphisms were detected successfully. This method that detects STR polymorphisms not only from fresh human fingernails, but also from old nail fragments stored at room temperature for up to 10 years is expected to become a novel DNA analytical method in forensic medicine and genetic studies.

Short Tandem Repeat Polymorphism of the D2S1242 Locus in a Japanese Population

Allele frequencies and sequence characteristics of the D2S1242 short tandem repeat (STR) locus were studied in a Japanese population sample. A total of 10 D2S1242 alleles and 34 genotypes were identified in 273 unrelated Japanese individuals. The five most common alleles detected had frequencies of over 10%. No deviations from Hardy-Weinberg equilibrium were found when the expected allele values were compared with the observed values. Sequence analysis of each allele showed a tetranucleotide polymorphism. Alleles 9 to 14 had different sequence structures than alleles 15 to 19. Allele 18 had a different sequence in the Japanese sample compared to an Austrian sample. The power of discrimination was 0.95. The present results demonstrate that the D2S1242 STR locus is a useful genetic marker in the Japanese population.

Individual identification by DNA polymorphism using formalin‐fixed placenta with whole genome amplification

Polymerase chain reaction (PCR) amplification using formalin‐fixed material is very limited. In the present study the use of 6 week formalin‐fixed placenta for individual identification was examined based on DNA analyses. The objective of the examination was to prove whether the placenta was from a woman who had just given birth. DNA extraction was carried out from the maternal blood sample and from the formalin‐fixed placental samples composed of three parts: maternal side, infant side and umbilical cord. One minisatellite (D1S80), 12 short tandem repeat (STR) polymorphisms and amelogenin X, Y were investigated. All the polymorphic systems were detected in the maternal blood sample. The majority of the DNA isolated from the placental tissues had molecular weights of approximately 500 bp, and only two to four STR loci were amplified using the DNA. In order to amplify more DNA polymorphic markers from the formalin‐fixed tissues, whole genome amplification was performed. After amplification by degenerate oligonucleotide‐primed PCR (DOP‐PCR), the products contained DNA with increased molecular weight up to >10 kbp. More DNA loci were typed using the DOP‐PCR products. Furthermore, large molecular size fragments were purified from the DOP‐PCR products by agarose electrophoresis, and then the D1S80 locus and 12 STR loci were successfully amplified using these fragments.

Frequency of D1S80 and HLA DQα alleles in a Chinese population

Allele frequency distributions for the D1S80 (MCT118) and HLA DQα loci were determined in a Chinese population sample using the polymerase chain reaction (PCR). A total of 25 alleles and 100 phenotypes were observed for D 1 S80. The frequency of allele 18 was higher than allele 24 only in this Chinese population when compared to other reported populations. A total of 6 alleles and 21 possible phenotypes were observed for HLA DQα. The power of discrimination was 0.97 and 0.93 for D1S80 and HLA DQα, 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.

Short Tandem Repeat Polymorphism in the Flanking Region of the Human Phosphoglycerate Kinase Gene in a Japanese Population

ABSTRACT The human phosphoglycerate kinase (PGK1) gene is located within Xq11–Xq13 and is closely linked to the androgen receptor gene within a region implicated in a number of X-chromosome-linked urologic disorders. A polymorphism of a TATC short tandem repeat (STR) is present downstream from the PGK1 3′ nuclease-sensitive site. We present the PGK1 flanking STR sequence and population genetic data for 190 Japanese males and 83 Japanese females. Ten STR alleles and 29 genotypes were identified in the population. Five alleles—*10, *11, *12, *13, and *14—were common in the Japanese with frequencies greater than 10%. No significant deviations from Hardy-Weinberg equilibrium were established. The power of discrimination was 0.993 for females and 0.819 for males; heterozygosity was 0.759 for females; and the polymorphic information content was 0.936. These data indicate that this STR locus shows a high degree of polymorphism in this Japanese population and may prove to be a useful genetic marker in forensic medicine, in determining the clonality of neoplasms, and potentially in studying predisposition to prostate cancer and other urologic diseases.

An Improved High Resolution Single Method for Orosomucoid ORM1 and ORM2 Phenotyping

An improved high resolution single method for orosomucoid ORM1 and ORM2 phenotyping using isoelectric focusing (IEF) in wide-scales ultrathin layer polyacrylamide gels of pH range 4.2 to 4.9 is presented. The method is reliable, simple, and provides an alternative for the three currently required ones for typing this genetic system.