| Objective: Short tandem repeat (STR) genetic marker system, which is widely distributed in human genome and has high genetic polymorphism and the fast test means, has been widely used in forensic practice and other fields. Autosomal STRs and mitochondrial DNA (mtDNA) genetic markers used in forensic medicine can solve most cases of identification discrimination and parentage testing. However, in many other forensic cases, DNA samples are highly degraded for many reasons, a loss of signal is typically observed with larger-sized STR products and it is difficult to have a complete genotype when using current commercially conventional multiplex STR kits. The analysis of the mitochondrial DNA (mtDNA) hypervariable regions is typically used, but its utilization is limited because it has a feature of haploid and maternal-only transmission and it is laborious and cost-prohibitive. MiniSTR assays, which is to reduce the size of the PCR products by moving primers as close as possible to the repeat region, produces a higher success rate for genetyping with degraded DNA samples. MiniSTR locus is definited as a new generation of genetic marker following STR by European DNA Profiling Group (EDNAP). The population genetic data of five miniSTR loci for D1S1677, D2S441, D4S2364, D10S1248 and D22S1045 have been reported in America, Japan and Span population, et al, while our country is lack of relative study and population genetic data. In the present study, we selected D1S1677, D2S441, D4S2364, D10S1248 and D22S1045 five miniSTR loci to investigate the genetic polymorphism in Han population of China and evaluated its application in forensic science.Methods:Genome DNA samples were extracted from whole blood of 115 unrelated healthy individuals of the Chinese Han population by using chelex 100. Forward primers of D1S1677, D4S2364, D10S1248 and D22S1045 were fluorescently labeled by 6-FAM in 5'end, while locus D2S441 was labeled by VIC. All DNA samples were amplified for five miniSTR loci. D2S441 and D10S1248 were multiplex amplified, while other loci were amplified singly. The PCR products were separated electrophoretically using an ABI3100 Avant Genetic Analyzer and the electrophoretic results were analyzed by using GeneScan 3.7 and Genotyper 3.7 software. Allele frequencies and genetic polymorphism parameters were calculated by counting method. Nine highly degraded DNA samples, which were not completely genotyped by commercial STR kit (Ampf/STR Identifiler, Applied Biosystems), were selected for detecting the five miniSTR genotype. These 9 samples consists of 4 blood samples from highly decomposed bodies, 3 low copy number DNA samples and 2 blood samples from eroded metal(from No.1~9). Then the samples were tested by miniSTR assays and the results were compared with that of the commercial kit tests.Results: Among the 115 whole blood samples, each locus was successfully genotyped, with the length of the amplicon less than 125bp. 7, 8, 5, 8, 8 alleles and 12, 21, 10, 15, 18 genotypes were respectively detected in the five miniSTR loci D1S1677, D2S441, D4S2364, D10S1248 and D22S1045. No deviation from Hardy-Weinberg equilibrium was observed in the five loci. The heterozygote observed (Ho) and polymorphism information component (PIC) were respectively 0.6609, 0.6957, 0.7478, 0.7652, 0.7826 and 0.5658, 0.7107, 0.5919, 0.7106, 0.7489 for D1S1677, D2S441, D4S2364, D10S1248 and D22S1045. The power of exclusion (PE) and the power of discrimination (PD) were 0.3530, 0.4824, 0.3637, 0.4901, 0.5023 and 0.7954, 0.8902, 0.8113, 0.8913, 0.9032 respectively for D1S1677, D2S441, D4S2364, D10S1248 and D22S1045. The accumulated power of exclusion and power of discrimination of the five miniSTR loci were respectively 0.9459 and 0.999953. The two multiplex amplified loci D2S441 and D10S1248 generated clear electrophoretic graphs and were successfully typed for all 115 samples. Complete genotype of the five miniSTR loci in 9 highly degraded DNA samples were successfully obtained, which demonstrated that miniSTR assay might have higher success rate than the conventional commercial STR kit.Conclusions: The Five miniSTR loci have better polymorphism which can be used for population genetics study and forensic medicine practice in Chinese Han population. The two loci D2S441 and D10S1248 are feasible to be multiplex amplified. The miniSTR assay has a higher success rate compared with commercial kit in genotyping degraded DNA samples and has a special value in forensic individual identification and paternity test for highly degraded samples. |
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