| Objective: With the diversity of criminal patterns and the augmentation of criminals'anti-detection awareness, it becomes more and more difficult to identify the suspect and make ture judgement on the casework. Therefore, how to resolve the identification of special biological samples, such as low copy DNA, highly degraded samples or mixture samples, has been a question that forensic scientists must face to. Short tandem repeat(STR) genetic marker system, which is widely distributed in human genome and has high genetic polymorphism, has been widely used in forensic paternity testing and individual identification because the allele size is short and easy to amplify. However, in some forensic casework, the DNA samples were highly degraded due to exposure to environmental elements or natural contaminants. These highly degraded samples were usually failure to type using the commercial multiplex STR kits, which showed either loss of information at higher molecular weight allel or loss of signal. MiniSTR assay technique, raised after 9.11 disaster, is able to reduce the size of the PCR products by moving primers as close as possible to the repeat region. As a result, it can be applied to examine highly degraded DNA samples and showed higher successful rate than common STR techonolgy. The population genetic data of five miniSTR loci D1S1677, D2S441, D4S2364, D10S1248 and D22S1045 have been reported in Japan, American and Span population. Three of these new markers(D10S1248, D2S441, and D22S1045) have been recommended for adding to Europe DNA database by European DNA ProfiLing Group (EDNAP) in 2005. However, our country is lack of relative study and population genetic data. In the present study, we selected D1S1627, D5S2500, D3S4529, D6S1017 and D9S2157 five miniSTR loci to investigate the genetic polymorphism in HeBei Han population and evaluated its application in forensic science.Methods:Genome DNA samples were extracted from whole blood of 125 unrelated healthy individuals of the Hebei Han population by using kit of TIAN GEN company. All DNA samples were amplified for five miniSTR loci. D3S4529, D6S1017 and D9S2157 were multiplex amplified, and D1S1627, D5S2500 were multiplex amplified. Forward primers of D6S1017and D3S4529 were fluorescently labeled by VIC in 5'end, D5S2500and D6S1017 by TAMRA, D9S2157 by FAM. The PCR products were separated electrophoretically using an ABI310 Genetic Analyzer and the electrophoretic results were analyzed by using GeneMapper3.2 software. Allele frequencies and genetic polymorphism parameters were calculated by counting method. To compare the effect of miniSTR and common STR, the blood was laied outside for 1 week to 8 weeks on summer day to model degraded samples, and then these degraded samples were tested by the 5 miniSTR multiplex typing system and the commercial commom STR kit. The DNA were extracted from the heart, kidney, liver, muscular tissue, skin and blood stain from the same corpse to test the tissue identity. The family survey were conducted using the samples from 10 pieces of parentage testing caseworks in our Center of Forensic Medicine Identification. The species-specificity was evaluated by detecting the PCR products of 5 miniSTR loci in the fish, pig, sheep and cow. 9947A DNA sample was diluted to 1ng, 0.5ng, 0.25ng and 0.125ng to test the sensitivity.ltiplex amplifn genetics study.Results: Two sets of fluorescence-labeled multiplex-PCR typing system for 5 miniSTR loci were established. Population genetic investigation results showed that 5, 5, 5, 6, 8 alleles were detected for the marker D1S1627, D5S2500, D3S4529, D6S1017 and D9S2157 respectively. The allele frequencies of the five miniSTR ranged from 0.0170~0.4260. 11, 13, 13, 15, 24 genotypes were respectively detected in the five miniSTR loci D1S1627, D5S2500, D3S4529, D6S1017 and D9S2157. The genotype frequencies of the five miniSTR ranged from 0.008265~0.347107. The loci showed no significicant deviations from Hardy-Weinberg equilibrium (P>0.05). The heterozygote observed (Ho) and polymorphism information component (PIC) were respectively 0.769, 0.795, 0.803, 0.725, 0.792, 0.790 for D1S1627, D5S2500, D3S4529, D6S1017 and D9S2157. The power of exclusion (PE) and the power of discrimination (PD) were 0.542, 0.590, 0.605, 0.468, 0.584 and 0.815, 0.867, 0.873, 0.900, 0.921 respectively for D1S1627, D5S2500, D3S4529, D6S1017 and D9S2157. The accumulated power of exclusion and power of discrimination of the five miniSTR loci were respectively 0.9835846 and 0.9999753. For the highly degraded DNA extracted from blood laid outside for 1 week, 2weeks, 4 weeks or 8weeks, the mini-STR assays resulted in complete DNA profiling in all of the 6 miniSTR loci, whereas the results of IdentifilerTM STR kit showed allele dropout or non alleles. The genotypes of the different tissues in the same cadaver were identical, indicating the tissue identity of the 5 loci. No mutation was found in the family survey. No specific amplified products were detected in some animals such as fish, pig, sheep and cow. 0.125ng, 0.25ng 9947A DNA template were successfully typed by the two sets of fluorescence-labeled multiplex-PCR typing system for 5 miniSTR loci,Conclusions: (1) In the present study, we obtained the population genetic data of five miniSTR loci D1S1627, D5S2500, D3S4529, D6S1017 and D9S2157 in Hebei Han Population. The Five miniSTR loci have better polymorphism which can be used for forensic personal identification and parentage testing. (2) We established two sets of fluorescence-labeled typing system for 5 miniSTR loci D1S1627, D5S2500, D3S4529, D6S1017 and D9S2157, which are valuable in the forensic identification, especially for the highly degraded samples, because of the higher successful rate than common STR typing technique, high sensitivity, species-specificity, tissue identity and genetic stability. |
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