| Objective: Fluorescent multiplex PCR system detecting short tandem repeats (STR) has been widely used in forensic paternity testing and individual identification. The two major commercial multiplex STR typing kits include IdentifilerTM kit and PowerPlex?16 kit, which can resolve most forensic cases of individual identification and trio paternity testing. However, the system efficiency of the two kits is not enough for the duo paternity testing and other deficient paternity testing. Therefore, the kits used for deficient paternity cases need to be further improved. In the present study, according to the literature reports, eight STR loci with high genetic polymorphism in Chinese Han Population were selected to construct a new multi-plex PCR system, including D7S3048, D8S1132, D11S2368, D2S1772, D6S1043, D13S325, D12S391, GATA198B05 and amelogenin locus. The sensitivity and species specificity of the system were tested. The population genetic data of 8 loci in Chinese Han population, Hui ethnic minority group, Tu ethnic minority group and Tibet ethnic minority group were investigated. In addition, this multi-plex PCR system was applied in several cases of motherless paternity testing combined with commercial STR kit to evaluate the value of the system in deficient paternity testing.Methods:DNA samples were extracted from FTA card of unrelated healthy individuals using Chelex 100. DNA was multiplex amplified for eight STR loci including D8S1132, D12S391, GATA198B05, D7S3048, D2S1772, D11S2368, D6S1043, D13S325 and a amelogenin locus. Forward primers of amelogenin, D8S1132 and D12S391 were fluorescently labeled by 6-FAM in 5'end, GATA198B05 and D7S3048 by HEX, D2S1772 and D11S2368 by TAMRA , D6S1043 and D13S325 by ROX. The multiplex amplification conditions such as the concentration of primer, Mg2+, Taq enzyme, anneal temperature, cycle times, and the time of pre-denaturation, final extension were optimized. The PCR products were separated electrophoretically using ABI310 Genetic Analyzer and ABI3130 Genetic Analyzer, and the electrophoresis results were analyzed using GeneMapper3.2 software. Two homozygotes in each locus were selected for sequencing, and the alleles in each locus were named according to the sequencing results and the principles of the international society of forensic haemogenetics (ISFG). The allele frequency of 8 loci in 200 Chinese Han individuals, 100 Hui individuals, 100 Tu individuals and 100 Tibet individuals were investigated,and the genetic polymorphism data were calculated and compared among different groups. The sensitivity and the species specific tests were performed for the STR multiplex systems. Ten affirmed paternity motherless parentage cases were analyzed using this eight STR multiplex system and commercial STR kit, and the combined PE and combined paternity index (PI) were calculated.Results:1 Development of the multiplex PCR system: the four fluorescense multiplex PCR system for eight STR loci and amelogenin was successfully constructed with clear genotyping graph and without non-specific amplification product peaks interfering DNA typing.2 The results of population genetics: No deviation from Hardy-Weinberg equilibrium was observed in all of the eight loci in the four populations (P>0.05). In Chinese Han population, Tibet ethnic minority group, Tu ethnic minority group and Hui ethnic minority group, the value of observed heterozygote (Ho) was respectively 0.803~0.905, 0.778~0.872, 0.720~0.900, 0.816~0.889, the value of the polymorphism information component (PIC) was respectively 0.770~0860, 0.750~0.860, 0.750~0.860, 0.750~0.860, the value of probability of exclusion (PE) was respectively 0.605~0.806, 0.558~0.739, 0.460~0.795, 0.630~0.773, the value of power of discrimination (DP) was respectively 0.927~0.967, 0.906~0.964, 0.913~963, 0.909~0.964, the value of matching probability (Pm) was respectively 0.033~0.073, 0.036~0.094, 0.037~0.087, 0.036~0.091, the cumulated power of discrimination was respectively 0.99999999998, 0.99999999996, 0.99999999996, 0.99999999996, and the combined power of exclusion was respectively 0.99989, 0.99993, 0.9998, 0.99993.3 Forensic Applications:3.1 The results of the sensitivity analysis demonstrated that 0.125ng DNA template could be successfully typed by this new STR multiplex amplification system.3.2 The study of species-specificity showed that among all of the detected non-primate animals (dog, rabbit, cow, fish, pig, chicken), primate animals (monkey) and common laboratory strains (Candida albicans, enterococcus , Escherichia coli), no specific peaks for the 9 loci was detected except for monkeys, in which there was only a peak in the amelogenin locus.3.3 Combined this system with the commercial kit in motherless cases,the cumulative probability of exclusion achieved to 0.9999, and the cumulative paternity index increased by 2 ~ 4 orders of magnitude, which greatly enhanced the reliability of identificaion conclusions in the motherless parentage cases.Conclusions: A multiplex PCR system for eight STR loci and amelogenin was successfully constructed with accurate, stable and reliable genotyping results and higher sensitivity (0.125ng), as well as human species specificity. The genetic polymorphism of the eight STR loci was quite high in Chinese Han, Hui, Tu, Tibet Population. This new multiplex STR system was valuable as a complement of commercial STR kit to resolving the deficient and difficult paternity cases.
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