DNA mixture has been a problem to be conquered for a long time in the forensic study. The DNA mixtures can be mainly divided into two categories: one comes from the sex assaults, for which we have to detect the sperms among the large amount of vaginal epithelial cells; the other one is the combination of different common samples, such as blood mixtures, salvia-blood mixtures and so on. In recent years, deeper and deeper study on DNA mixtures has led a way to objectiveness and pluralism. Novel techniques, like fluorescence- or magnetic-activated cell sorting strategies,micromanipulation and acoustic different analysis can be utilized to separate sperms in the mixtures; as for the second sort, the application of massively parallel sequencing(MPS), microfluidic droplet, whole-genome amplification, emulsion PCR(ePCR) et al. rise up the chances to detect the minor contributor in the mixtures. Furthermore, the development of both traditional and novel biomarkers which includes STR, Y-STR, SNP, DIP, Microhaplotype, DIP-STR, SNP-STR,, mtDNA-SNP and so on, provide us more analyzing choices in mixture study. The last part of the assay focuses on the latest progresses of evaluating the number of contributors, the explanation theory and calculation software.

Objective To observe anomalous genotypes of 4 multi-copy RM Y-STRs in Han population in Hubei province. Methods 252 unrelated male samples were ampliifed using reported and newly designed primers, then detected and analyzed by AB 3130 genetic analyzer. Results A total of 25 anomalous multi-band patterns were observed in 20 samples corresponding to an incident rate of 7.94%. 5 anomalous genotypes were observed in DYF387S1 locus, 15 in DYF399S1, 1 in DYF403S1 and 4 in DYF404S1. Four samples showed extra alleles in more than one locus. Conclusion Anomalous genotype has high incident rates in RM Y-STR markers and requires extensive attention in forensic practice.

In recent years, the cases of prenatal paternity testing gradually increased in forensic practice. The traditional prenatal paternity analysis can be performed only after invasive sampling of chorionic villi or amniotic fluid, which can result in a risk of miscarriage. The existence of circulating cell-free fetal nucleic acid in maternal plasma has brought new opportunities for the noninvasive prenatal paternity testing. In this paper, the research situation and application prospect of circulating cell-free fetal nucleic acid in maternal plasma in prenatal paternity testing are reviewed.

Forensic medical talents with creative ability should have ability to find.analyze and solve problems from complex cases.According to teaching practice on the basis of PBL this article illustrates application methods of PBL teaching characters in the training of forensic medical talents with creative ability.

Three SNaPshot multiplex assays were developed to test 23 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable regions (HVR)I and II, which was aimed at increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and confirming haplogroup assignments of mtDNA profiles in both human population studies and medical research. The selected SNPs targeted the East Asian phylogeny. These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly. The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 31 haplotypes, and the haplotype diversity was estimated to be 0.952. The multiplex SNaPshot method is rapid and robust, and suitable for large-scale screening studies of mtDNA variability.

Three SNaPshot multiplex assays were developed to test 23 coding region single nucleo-tide polymorphisms (SNPs) and one control region SNP outside hypervariable regions (HVR) Ⅰ and Ⅱ, which was aimed at increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and confirming haplogroup assignments of mtDNA profiles in both hu-man population studies and medical research. The selected SNPs targeted the East Asian phylogeny. These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly. The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 3 i haplotypes, and the haplotype diversity was estimated to be 0.952. The multiplex SNaPshot method is rapid and robust, and suitable for large-scale screening studies of mtDNA variability.

Objective To develop a simplified bisulfite genomie sequencing(BGS)method for DNA methylation marker scanning.Methods According to modified BGS protocol,the desalt DNA treated with bisulfite were directly used for bisulfite-PCR(BSP)without alkali treatmenL Complement of the bisulfite modification Wag accomplished by a prolonged pre-denaturation stage.After BSP,a second round PCR was performed with a pair of GC tagged primers to adjust the GC content of the amplieon for direct sequencing.To assess this improved protocol,promotor methylation of TNF-α gene in 3T3-L1 cell and androgen receptor(AR)gene in Hela cell was investigated.The real time BSP for Alu was also used to compare the sensitivity of the modified assay with traditional assay.Results Both the hypermethylated TNF-α promotor and hypomethylated AR promotor were successfully sequenced by improved BGS method,and the results were consistent with that of the traditional assay.The conversion rate reached 100%,while the conversion specificity was higher than 93.75%.The sensitivity of improved BGS method inereaged significantly(t=2.978 2,P＜0.05)and showed good reproducibility.Condusion The improved BGS method is simple and sensitive,facilitating more ambitious genomic methyhtion mapping 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 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.

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.