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Objective: To evaluate the forensic utility of Y-single nucleotide polymorphisms (SNPs) markers., Methods: Allele-specific PCR, restriction enzyme digestion or direct PCR were performed to examine 10 different SNP loci on Y chromosome, namely M9, M15, M45, M89, M95, M122, M134, M145, M173 and P25 in 161 Chinese Han males., Results: A total of 8 of the 10 SNPs are reported to be polymorphic in Chinese. The gene diversity for the loci showing polymorphism ranged from 0.988/0.012-0.752/0.248, with a power of discrimination 0.094-0.373. Loci M122 and M134 were the most polymorphic markers in Chinese Hans. Nine different haplogroups with frequencies from 1.2% to 51.6% were observed and 3 of the haplogroups-K*(x O2a, O3, P), O3*(x O3e) and O3e were found in 75.2% of Chinese Hans., Conclusion: A comprehensive gene diversity data of Y chromosome and haplogroups were obtained in Sichuan Han population, which will be served as the base for using these Y-SNP markers in forensic medicine and individual identification in Sichuan Hans.

Objective: To formulate recommendations in the evaluation of results of genetic analyses in paternity testing under considering mutations., Methods: A total of 15 short tandem repeat(STR) loci were employed for this study, which were included CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, PentaD and PentaE. Both 100 cases of true trio and 100 cases of false trio were investigated., Results: The numbers of mismatch alleles in different STR loci were observed in 100 cases of false trio. The different distributions of paternity index were obtained, including the changes of paternity index in each case of true trio under simulated mutations., Conclusion: In order to avoid the effect of mutations, the exclusion of paternity was never considered on the basis of a single locus. The threshold values of the combined probability of exclusion and the paternity index were important for both exclusion and inclusion of paternity. The scientific evidence for paternity testing can be obtained when both the combined probability of exclusion and the paternity index meet the threshold values. However, when either the combined probability of exclusion or the paternity index can not meet the threshold values, more genetic markers should be added.

Objective: To develop a multiplexed mutagenically separated PCR (MS-PCR) for single nucleotide polymorphism (SNP) loci typing in mitochondrial DNA coding regions and to study the applications in investigating the allele frequencies and haplotypes of four SNP loci in mitochondrial DNA coding regions in Chinese Chengdu Han population., Methods: Four SNP loci C12705T, A8701G, G8584A and C10400T, two allele specific forward primer with 4 bases different in size and a common reverse primer were designed for SNP typing. The primers simultaneously were amplified in a single tube. The genotyping of SNPs was determined by the two allele specific fragments different in size after polyacrylamide gel and silver staining., Results: The different SNP loci comprised a single band with different size respectively. Typing results were completely consistent with those by direct sequencing. The allelic frequencies of C12705T, A8701G, G8584A and C10400T were 0.3813/0.6187, 0.4813/0.5187, 0.8250/0.1750 and 0.4938/0.5062 respectively. A total of 6 different haplotypes was identified and the genetic diversity reached 0.7137., Conclusion: Multiplexed MS-PCR is a simple, rapid, accurate and efficient method for SNP typing, which will be very powerful for SNPs in the database establishing of mitochondrial DNA coding regions, the testing of forensic and population genetics research.

Objective: To obtain the data in polymorphism distribution of the five short tandem repeat (STR) loci: D18S979, D11S2014, D18S548, D1S1667 and GATA164F07 of Chinese Han population in Chengdu, and to evaluate their usefulness in the field of species specificity in forensic science., Methods: PCR, polyacrylamide gel electrophoresis (PAGE) and silver staining techniques were used to analyze the DNA samples from 100 unrelated individuals of Chinese Han ethnic group in Chengdu. Twelve different animals: monkey, pig, dog, bull, goat, chicken, duck, eel, mudfish, rabbit, guinea pig and mouse were selected as controls in this study for evaluating the species specificity of the five STR loci., Results: Six alleles and twelve genotypes were observed in D18S979. Five alleles and eleven genotypes were observed in D11S2014. Five alleles and thirteen genotypes were observed in D18S548. Seven alleles and nineteen genotypes were observed in D1S1667. Six alleles and fourteen genotypes were observed in GATA164F07. The genotype distributions of the five loci were analyzed by some related software and no deviation from the Hardy-Weinberg equilibrium was observed. Evaluated by way of using different animals as controls, monkey had amplification products at the extra-typing field of D18S979, D11S2014 and D1S1667. Bull, dog and eel had amplification product at typing field of D18S979, and pig, duck, mouse and rabbit had weak product. Bull had weak product at the typing field of D18S548. Dog, goat and eel had product at the typing field of D1S1667. Dog had weak product at the typing field of GATA164F07. Mudfish, chicken and guinea pig had no amplification product at the five loci., Conclusion: These data indicate that D18S979, D18S548, D1S1667 and GATA164F07 are highly polymorphic and D11S2014, D18S548 and GATA164F07 can play a key role in species identification.

Objective: To obtain the data in polymorphism distribution of the seven short tandem repeat (STR) loci: D1S2142, D1S3733, D2S1774, D3S2459, D21S1409, D21S1437 and D21S2055 of Chinese Han population in Chengdu, and evaluate the polymorphism data usefulness to the forensic science., Methods: PCR, polyacrylamide gel electrophoresis (PAGE) and silver staining techniques were used to analyze the DNA samples from unrelated individuals of Chinese Han ethnic group in Chengdu., Results: Eleven alleles and twenty-three genotypes were observed in D1S2142. Eight alleles and nineteen genotypes were observed in D1S3733. Eight alleles and fifteen genotypes were observed in D2S1774. Seven alleles and nineteen genotypes were observed in D3S2459. Six alleles and twelve genotypes were observed in D21S1409. Nine alleles and twenty-six genotypes were observed in D21S1437. Twenty alleles and seventy-seven genotypes were observed in D21S2055. The genotype distributions of the seven STR loci showed no deviation from the Hardy-Weinberg equilibrium. The parentage testing of 50 cases revealed an autosomal codominant inheritances and no mutations happened to seven STR loci., Conclusion: These data indicate that D1S2142, D1S3733, D2S1774, D3S2459, D21S1409, D21S1437 and D21S2055 have good polymorphism, with high probability of exclusion and probability of discrimination power as well as being loci available as the candidate genetic markers to forensic parentage testing and personal identification.

Objective: To understand the role of mitochondria associated signaling pathway in the apoptosis of human vascular endothelial cell induced by homocysteine (Hcy)., Methods: The mRNA and protein expression levels of the up-stream signaling molecules of caspase 3, Bcl 2, caspase 9, and cytosolic cytochrome-c, were investigated. The in vitro cultured human umbilical vein endothelial cells with homocysteine at different concentrations were incubated for 24 h. The expressions of Bcl 2 and caspase 9 at mRNA and protein levels were analyzed by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. Cytochrome-c in cytoplasm was also detected by Western blot., Results: The expression levels of three signaling molecules were all down-regulated by homocysteine at both mRNA and protein levels in a dose-dependent manner., Conclusion: Homocysteine could affect the formation of apoptosome through repressing the expression of Bcl 2 gene and release of cytochrome-c from mitochondria. Decreasing of apoptosome could disturb the activation of caspase 9. The results also indicate that the mitochondria pathway is not the major signaling pathway involved in Hcy-induced apoptosis.

Objective: To solve the problems in the accuracy and standardization of short tandem repeats-polymerase chain reaction (STR-PCR) typing, the authors adopted the molecular clone technology in producing the standard allelic ladders of D1S1676, D2S2735, D11S1977 and D22S444 loci and applied them in a population study on the Hans in Chengdu, China., Methods: PCR was used to produce several different allelic fragments of these loci. PCR products were eluted from the gel and re-amplified by PCR. The purified allelic fragments were then blunt-end subcloned individually into the pGEMR-T plasmid vectors and the recombinant were transfected into competent E.coli DH5alpha TM cells. The results of sequencing confirmed that the size and the construction of the inserts were correct. The recombinant plasmids DNA with the inserts were then used as template for re-amplification to generate the four loci standard ladders., Results: The authors succeeded in producing large quantity of standard allelic ladder of these four loci, with which the genetic polymorphisms of these loci in Chengdu Han population of China were studied., Conclusion: This method is of high value for forensic DNA typing to construct standard ladders. D1S1676, D2S2735 loci are robust for forensic analysis in Chinese Han population, whereas the value of D11S1977 and D22S444 loci is limited.

Objective: To develop a set of new markers for forensic application, the authors have chosen 6 short tandem repeat(STR) loci to study the allele frequencies and species specificity in Chinese Han population in Chengdu., Methods: One hundred and ten EDTA-blood samples were collected from the unrelated individuals in Chengdu city, Sichuan province. DNA was extracted by Chelex-100 and amplified by the polymerase chain reaction(PCR). Polyacrylamide gel electrophoresis (PAGE) and silver staining were used to analyze the PCR products., Results: The polymorphisms of all 6 STR loci have been obtained in Chinese Han population in Chengdu, the alleles of D4S2366, D4S2367, D6S474, D6S1281, D2S1396 and D20S601 being 7, 7, 6, 7, 5, 7, the observed heterozygosity of them being 0.802, 0.708, 0.770, 0.627, 0.542, 0.672, the discrimination power of them being 0.887, 0.828, 0.849, 0.848, 0.794, 0.865; and the power of exclusion of them being 0.602, 0.441, 0.544, 0.325, 0.227, 0.386. Evaluated by comparison with the data from 14 different animals as controls, the 6 STR loci contain good specificity of human beings., Conclusion: The 6 STR loci are highly polymorphic and can play a key role in species identification. They are new candidate markers for forensic personal identification and paternity testing.

Objective: To establish a simplified method of multiplex PCR based on chimeric primers for STR loci., Methods: A set of chimeric primers and universal primers were designed to carry out a multiplex PCR for amplifying four short tandem repeats(STR) loci. Then the amplified STR loci were detected by polyacrylamide gel electrophoresis and the gels were sliverstained., Results: A quadriplex STR system was developed on the basis of both chimeric and universal primers., Conclusion: The STR multiplex PCR based on both chimeric and universal primers hss been achieved readily and reproducibly by simple adjustment of the individual primer concentrations. The use of chimeric primers provides a method for primer design that eliminates the multiple optimization steps involved in developing the multiplex PCR.

Objective: In Caucasian population, the most common molecular basis for C8 beta deficiency s a single C to T transition in exon 9 of C8 beta gene resulting in a stop codon. In previous family studies, two individuals were identified with C8 beta complete deficiency and were found to be only heterozygous for this mutation. This study was conducted by the present authors in search of other possible causes for these two C8 beta deficient individuals., Methods: Using direct DNA sequence analysis of all exon-specific PCR products of the C8 beta gene from these two C8 beta deficient patients and their descendants., Results: Two other C to T transitions at base 298 and 388 in exon 3 were detected, which could also create a termination codon. The descendants from one of the deficient patients were also analysed for the mutations, and it could be demonstrated that the two C to T mutations in exons 9 and 3 are segregating independently., Conclusion: These two mutations, which create a termination codon, are sufficient to explain the complete C8 beta deficiency in both patients.