| Objective: Mini short tandem repeat (STR) technology, which could reduce the size of PCR products by moving the primers as close as possible to the STR repeat region, had been applied to examine highly degraded DNA samples and showed higher successful rate. Many studies confirmed that miniSTR technology was a valid method for highly degraded DNA samples. In 2006, the corporation of Applied Biosystems developed the first commercial miniSTR kit named MiniFilerTM and it had been used in practical case analysis. However, there was only eight STR loci belong to combined DNA index system (CODIS) included in the MiniFilerTM kit, and the system efficiency of it was not enough to perform individual identification and paternity testing. Therefore, to explore more miniSTR loci for forensic application and produce miniSTR kit by our country, 26 non-CODIS miniSTR loci reported by Hill et al had been studied in our lab since 2005. According to the population genetic data of the 26 miniSTR loci in Han population of China, 12 loci with high polymorphism were selected to develop miniSTR multiplex systems. Two miniSTR multiplex typing systems had been established containing 8 loci. This study was to establish the third miniSTR multiplex system including D6S474, D20S482, D4S2408 and D6S1017 four loci. To assure the reliability of genotyping, the standard allelic ladder was made by molecular cloning technology. In addition, the genetic polymorphism of four loci in Han and Hui population of China was investigated using this system and the application in forensic cases was evaluated.Methods:Genome DNA samples were extracted from whole blood of 135 and 114 unrelated healthy individuals of Han and Hui population using Chelex-100. DNA templates were multiplex amplified for D6S474, D20S482, D4S2408 and D6S1017 four miniSTR loci by fluorescence-labeled multiplex-PCR technology, and the PCR products were separated by capillary electrophoresis on ABI 310/3130 Genetic Analyzer. Fragment size and genotype were analyzed using GeneMapper ID Version 3.2 software. The multiplex amplification conditions were optimized according to the outcomes with the different primer concentration, Mg2+ concentration, DNA amount, annealing temperature and cycle times. All DNA samples were genotyped for the four miniSTR loci using the optimized miniSTR multiplex typing system.The standard allelic ladder was constructed using molecular clone technique according to the population genetic results. Each allele in each locus was named in line with the sequencing results and the principles of the international society of forensic genetics (ISFG). Genotypes of all DNA samples were typed and named on the basis of the standard allelic ladder. The allele frequencies and genetic polymorphism parameters were calculated to evaluate the genetic polymorphism of the four miniSTR loci.A series of validation experiments, such as the sensitivity test and the application in analyzing highly degraded DNA samples were performed with the miniSTR multiplex system and the commercial STR kit to compare the successful rate of them.Results: A fluorescent multiplex miniSTR system and corresponding allelic ladders for four miniSTR loci was successfully developed. Investigation of population genetics using the above multiplex typing system showed that 7, 8, 6, 6 alleles and 14, 18, 14, 14 genotypes were respectively detected in D6S474, D20S482, D4S2408, D6S1017 locus in 135 unrelated healthy individuals of Han population; 6, 7, 5, 7 alleles and 15, 23, 13, 17 genotypes were respectively detected in 114 unrelated healthy individuals of Hui population. No deviation from Hardy-Weinberg equilibrium was observed in the four loci. The value of the observed heterozygote (Ho) for D6S474, D20S482, D4S2408, D6S1017 locus was 0.748, 0.711, 0.748, 0.659 in Han population, which was respectively 0.759, 0.706, 0.714, 0.804 in Hui population. The value of polymorphism information component (PIC) was respectively 0.68, 0.66, 0.67, 0.64 in Han population, which was respectively 0.70, 0.73, 0.69, 0.68 in Hui population. The vale of power of exclusion (PE) was respectively 0.507, 0.446, 0.507, 0.368 in Han population, which was respectively 0.525, 0.438, 0.451, 0.606 in Hui population. The value of discrimination power (DP) was respectively 0.874, 0.871, 0.862, 0.851 in Han population, which was respectively 0.884, 0.911, 0.880, 0.860 in Hui population. The accumulated PE and cumulated DP of the four miniSTR loci were respectively 0.914902 and 0.999666 in Han population, which were 0.942257 and 0.999827 in Hui population. The results of the sensitivity analysis demonstrated that 0.0625ng DNA template could be successfully typed by the fluorescent multiplex miniSTR system for four miniSTR loci. For the highly degraded DNA extracted from blood layed outside for five months or digested by DNase I for 25min, the miniSTR assays resulted in complete DNA profiling in all of the four miniSTR loci, whereas the results of commercial STR kit showed allele dropout or non alleles.Conclusions: A fluorescent multiplex miniSTR system for four miniSTR loci including D6S474, D20S482, D4S2408 and D6S1017 was established and the relevent standard allelic ladders were developed. The investigation of genetics of Han and Hui populations using the above system showed that the four miniSTR loci have high genetic polymorphism. The typing system showed higher sensitivity (0.0625ng) and stability, as well as higher successful rate for analyzing the highly degraded samples than the common STR technique. In conclusion, this miniSTR typing system not only is valuble in the forensic identification of highly degraded samples, but also can be added to the common STR system to increase the discrimination power and the efficiency of the system. |
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