Objective To screen polymorphic biallelic markers on human Y chromosome in Han population , calculations of their allele and haplogroup frequency distributions to provide data for forensic application and population evolution studies. Methods Genotyping of 8 biallelic markers on human Y chromosome (M9, M89, M111, Ml19, M122, M134, IMS-JST0033050 and SRY +465) were carried out in a sample of 160 unrelated Chinese male individuals living in Wuhan using fragment length discrepant allele specific PCR (FLDAS-PCR) and PAGE technique. Results Genetic polymorphism were identified for all 8 biallelic markers in Wuhan Han population. Gene Diversity (GD) ranges from 0.0126 to 0.4830. A total of 9 different haplogroups(Hg 1-9)were observed and the haplogroup diversity (HD) were 0.7776. Conclusion The haplogroups formed by 8 biallelic markers are highly polymorphic, and can be used in conjunction with Y-STRs in forensic medicine and population evolution studies.

Objective To develop a PCR-based method of detecting plankton 16S rDNA for the i dentification of death by drowning. Methods Fifteen Sprague-Dawley rats were divided randomly into three groups: the death by drowning group, the group of submerging after death and the control group. After sacrificing by different ways, the brain, liver, kidney and lung of rats were taken out respectively and DNA were extracted from the tissues of these organs and were amplified subsequently by specific primers selected from the third and fourth variable regions of plankton 16S rDNA. Results The specific amplification products were detected from all 5 samples of lung tissue ( 100% ) , 4 samples from liver and kidney tissues (80% ) , and 3 samples from brain tissue (75% ) in the group of death by drowning. No amplification product was detected in all samples of the control group and the amplification product was detected only in 1 sample of lung (20% ) in the group of submerging after death. Conclusion The PCR-based method of detecting plankton 16S rDNA for the identification of death by drowning is certainly feasible.

Objective To investigate age-dependent variation of 5-methylcytosine (5-mC) content in human peripheral leukocytes.Methods 94 healthy individuals (50 males and 44 females) of ages varying from 4 to 87 years were dividied into six groups by age: age under 20 years, age between 20 to 30 years, age between 30 to 40 years, age between 40 to 50 years, age between 50 to 60 years and age beyond 60 years. Their 5-mC content in human peripheral leukocytes were analyzed by HPLC.Results The 5-mC content of age between 50 to 60 years (mean age 54.7) and age beyond 60 years (mean age 71.7) are statistically not significant, but statistically highly significant (P

Objective To study the primer design for Penta D and Penta E genotyping and investigate the genetic polymorphisms of these two loci in Chinese Han population in Wuhan.Methods 281 unrelated Chinese individuals living in Wuhan were typed by hot-start PCR with re-designed Penta D and Penta E genotyping primers and PAGE technique,and the results were compared with those by the PowerPlex TM 16 system of Promega.Results The amplified fragment size of Penta D and Penta E loci with re-designed primers ranged between 153～198bp and 107～212bp respectively,which were consistent with the results obtained by the re-designed primers and PowerPlex TM 16 system,and the re-designed primers had a higher sensitivity than PowerPlex TM 16 system (0.2ng vs 0.5ng) by silver staining.10 and 21 alleles were observed for Penta D and Penta E in Chinese Han population in Wuhan,and the genotype distributions of the two loci were in accordance with Hardy-Weinberg equilibrium.Family studies confirmed Mendelian inheritance of alleles.The power of discrimination (DP) for Penta D and Penta E were 0.926 2 and 0.986 0,and the power of exclusion (PE) were 0.665 1 and 0.832 5 respectively.Conclusion The re-designed primers for Penta D and Penta E genotyping are reliable.These two loci are highly polymorphic in Chinese Han population and can be used in forensic identification and paternity test.

Objective To study the Application of X-linked differentially methylated polymorphism site in forensic medicine.Methods X-STR HUMARA was chosen as a model locus.PCR procedures were performed after digestion using methylation-sensitive restriction endonucleases.STR polymorphism of HUMARA was analyzed and compared in samples collected from male and female individuals.Result After digestion with methylation-sensitive restriction enzyme HpaⅡ,no PCR products were obtained from male samples,whereas PCR products from the female samples were normally typed.In monoclonal tumor cell samples from females,only one allele was detected.Conclusion The differentially methylated X-STR HUMARA locus is a novel marker for mixture analysis of mixed stains,sex determination and discrimination of tumor tissues.

Drowning represents one of the leading causes of unnatural death. The plankton test is still considered as one of the useful methods for medico-legal investigation of death by drowning although controversies concerning the reliability of the test exist. The methods commonly used for detection of planktons generally include microscopic examination after tissue samples are destructed with or without strong acid, and detection of the planktons by molecular biological techniques. The evaluation of the positive results by the former methods are limited by the physical-chemical characters of the planktons. Detection of DNA genetic markers by molecular biological techniques, which can provide abundant information and is applicable to studying various planktons, is a novel method helpful for investigation of drowning. In the present paper, several techniques for detection of planktons were reviewed as references for medico-legal practitioners in investigation of suspected cases of drowning.

Objective To evaluate the value of detecting chlorophyll related genes of plankton in the diagnosis of drowning. MethodsEighteen rabbits were divided randomly into three groups: death by drowning (n=10), postmortem submersion (n=6) and control (n=2). The heart blood, lung, liver, kidney and brain tissues were taken from every rabbit. After isolated plankton from tissues with percoll and extracted their DNA, the chlorophyll-related genes, including EG (EG1 and EG2) and SK (SK1 and SK2), were detected using PCR technique. Meanwhile, diatom test was also performed from lung and liver tissues by nitric acid digestion method. ResultsFor the drowning group, the specific amplification products for EG1 were detected from 9 samples in heart blood, 10 samples in lung, 9 samples in liver, 7 samples in kidney and 8 samples in brain. The products for EG2 were detected from 8 samples, 10 samples, 7 samples, 5 samples and 7 samples accordingly. There were a small number of positives in heart blood, lung and kidney with SK1 and SK2 (≤2). For the postmortem submersion group, only one case was positive from heart blood and lung tissue respectively for EG1. No amplified product was detected for EG1 and EG2 in various tissues in control group, and also no product was detected for SK1and SK2 in other groups. In addition, diatoms were detected from 9 lung and 3 liver tissues in drowning group with the nitric acid digestion, and only one sample of lung was positive in the postmortem submersion group. ConclusionThe detection rate of the chlorophyll-related gene EG with PCR method was higher than that of diatom with nitric acid digestion method in drowning victims, and it can be used as a potentially useful tool for diagnosing drowning.

Objective To establish a simple and high-performance analytical technique for detecting DNA methylation markers and SNPs simultaneously,and obtain the population genetics data of some SNPs in the hypermethylated region upstream of the human H19 gene.Methods The haplotypes of H19FR1 and H19FR2 which located in the promoter region upstream of the human H19 gene were investigated from 232 unrelated Chinese individuals living in Wuhan by means of PCR and subsequent denaturing gradient gel electrophoresis(DGGE).Based on the methylation status of the genomic DNA,selective detection of the parental alleles for H19FRs was examined by using the methylation-sensitive restriction enzyme(msRE) Hpa II or Hha I.Results Five haplotypes and nine phenotypes were observed for H19FR1 in Chinese Han population in Wuhan,and the power of discrimination(DP),polymorphism information content(PIC) and probability of paternity exclusion(PE) were 0.803,0.58 and 0.322 respectively.For the H19FR2,two haplotypes and three phenotyes were detected,and the DP,PIC and PE were 0.626,0.37 and 0.162 respectively.Sequencing results showed that there were 3 SNPs,a7342g,a7357g and g7547a,and one g7351c point mutation in H19FR1.In the H19FR2,there was only one SNP,a8097g.The msRE,HpaⅡ or HhaⅠ,could digest the maternal allele,and only a single band derived from the paternal allele was detected by post-digestion PCR-DGGE(PDP-DGGE) technique.Conclusion PDP-DGGE is a simple,sensitive and effective technique for analyzing DNA methylation status and SNPs simultaneously,and can be used for discriminating the parental origin of alleles.

Objective Study on the pattern of changes of TGF-?1 and type I receptor occurred in the experimental fluid percussion brain injury model for the purpose of providing the scientific basis for molecular pathological diagnosis, forensic identification, clinical treatment as well as further ascertaining the molecular mechanism of brain injury. Methods Male Sprague-Dawley rats were divided into normal control, sham operation control and injury groups. The rats of injury groups were subjected to moderate lateral fluid percussion brain injury (0.2 MPa). The injury groups were then subdivided into 30min, 1h, 3h, 6h, 12h,1d, 3d and 7d sub-groups according to the time elapsed after injury. Immunohistochemistry SP method was used for studing the immunoreactivity of both TGF-?1 and T?R I factors. Results (1) In the brain of normal control and sham operation control groups, the low expression levels of TGF-?1 and T?R I were observed; (2) The gradual increase of TGF-?1 and T?R I immunoreactivity could be observed 1 to 3d after injury both in cortex and brain stem, and sustained at the high level up to 7d; (3) In hippocampus, the gradual increase occur during 12h to Id after brain injury, and sustained the high level at 3d, then declined at 7d. Conclusion The results suggested that brain injury induced the gene eypressions of the TGF-?1/ T?R I . The TGF-?1/ T?R I may contribute to maintance of nerve cell survival and the repair of damaged neural tissues after CNS injury and the patterns of their level change were quite regular and can be used for timing of injury in forensic medicine aspect.

Objective Study on the pattern of changes of bFGF and FGFRl mRNA occurred in the experimental brain injury model in order to provide scientific basis for the diagnosis, forensic identification and clinical treatment, and also for further ascertaining the molecular mechanism of brain injury. Methods Male Sprague-Dawley rats were divided into 3 groups: normal control, sham operation, and injury groups. The rats of injury groups were subjected to moderate lateral fluid percussion brain injury (0.2MPa). The injury groups was then subdivided into 30min, 1h, 3h, 6h, 12h, 1d, 3d and 7d groups according to the time elapsed after injury. In situ hybridization (ISH) and RT-PCR were used for studying the mRNA expression of both bFGF and FGFRl factors. Results (1) In the brain of normal control and sham operation control groups, mRNA levels of bFGF and FGFRl were low; (2) There is gradual increase of bFGF and FGFRl mRNA levels could be observed 6h to 3d after injury both in cortex and brain stem, then partly declined at 7d; (3) In hippocampus, the gradual increase occurred during 3h- 1d after injury, then partly declined at 3d, and returned to basal level at 7d. Conclusions The results suggested that brain injury induced the gene expressions of bFGF and FGFR1. The bFGF may contribute to maintenance of nerve cell survival and the repair of damaged neural tissues after CNS injury and the patterns of their level change were quite regular. It is potentially useful for timing of injury in forensic medical practice.