Construction Of Four Color Fluorescence-Labeled Multiplex Typing System For Six MiniSTR Loci D9S1122/D10S1435/D12ATA63 And D20S1082/D18S853/D17S1301 And Evaluation Of Its Forensic Application

Objective: Short tandem repeats (STR), also named microsatellite DNA, is a kind of genetic marker consisted of 1~6bp tandem repeats. Most of the STR loci locate in the non-coding regions of human genome, and the few is in the coding region. STR has been studied and applied in many fields such as phymatology, anthropology, hereditism, and forensic science, etc., because of its extensive distribution in the genome, high polymorphism and simple analytic methods. At present, STR has been used as a helpful tool for forensic science to solve personal identification and parentage testing. However, it is usually difficult to get complete or correct genotype for many highly degraded DNA samples in forensic cases using current commercially conventional mutiplex STR kits. Whereas miniSTR assay technique, which is to reduce the size of the PCR products by moving primers as close as possible to the repeat region, produces a higher successful rate for degraded DNA samples'genotyping. MiniSTR loci are defined as a new generation of genetic marker following STR by European DNA profiling Group (EDNAP). In 2006, the corporation of Applied Biosystems developed a commercial miniSTR kit named MiniFiler consist of eight CODIS loci with shorter PCR products. The population genetic data of five non-CODIS miniSTR loci for D1S1677, D4S2364, D10S1248 have been reported in America, Japan and Span population, while our country is lack of relative study and population genetic data. Presently most studies about miniSTR were limited in the CODIS system. But not each of the CODIS loci can be designed to be miniSTR. Because the range of allele length is too long or the locus is not fit for designing new primers in flanking sequence, it is difficult to make those loci decrease to ideal length (<150bp). In the present study, to resolve the identification of the highly degraded samples, we selected six non-CODIS minSTR loci D9S1122, D10S1435, D12ATA63, D20S1082, D18S853 and D17S1301 to establish fluorescence-labeled multiplex typing system and investigate the allele frequency in the Han population in Hebei Province, as well as evaluated the value of forensic application of the system.Methods: Genome DNA samples were extracted from whole blood of 115 unrelated healthy individuals of the Hebei province Han population by using TIANGEN blood genome DNA extraction kit. The samples of family survey were come from parentage testing caseworks in our Center of Forensic Medicine Identification. The samples of tissue identity were from the same corpse. The genome DNA of the above samples was extracted using Chelex-100 method. In addition, the blood was layed outside for 1 week to 8 weeks on summer day to model degraded samples using two-step Chelex-100 method to extract DNA. The sense primer of D9S1122 and D20S1082 were labeled with HEX fluorescence, D12ATA63, D17S1301 with 6-FAM fluorescence, D10S1435, D18S853 with TAMRA fluorescence. D9S1122,D10S1435, D12ATA63 and D20S1082, D18S853, D17S1301 loci were multiplex amplified and the amplified products were separated by capillary electrophoresis. The data was collected by 310 data collection software and analyzed by Genemapper 3.2 software. The allele frequency and genetic parameters were calculated using PowerStatsV1.2 and GenAlEx6 software.Results: Two sets of fluorescence-labeled multiplex-PCR typing system for 6 miniSTR loci were established. Among the 115 whole blood samples, 8, 7, 5, 8, 6, 7 alleles and 22, 23, 12, 18, 15, 17 genotypes were respectively detected in the D9S1122, D10S1435, D12ATA63, D20S1082, D18S853, D17S1301 locus. The heterozygote observed (Ho) and polymorphism information component (PIC) in D9S1122, D10S1435, D12ATA63 and D20S1082, D18S853, D17S1301 locus were respectively 0.736, 0.806, 0.841, 0.776, 0.765, 0.767 and 0.720, 0.750, 0.630, 0.700, 0.670, 0.690. The power of exclusion (PE) and the power of discrimination (PD) were 0.486, 0.609, 0.677, 0.571, 0.555, 0.540 and 0.894, 0.900, 0.706 0.855, 0.858, 0.848. The accumulated power of exclusion and power of discrimination of the six miniSTR loci were respectively 0.994299 and 0.999990. The genotypes of the different tissues in the same cadaver were identical, indicating the tissue identity of the 6 loci. No specific amplified products were detected in some animals such as rabbit, fish, pig, and chick. No mutation was found in the family survey. 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.Conclusions: In the present study, we constructed two sets of four color fluorescence-labeled typing system for 6 miniSTR loci D20S1082/D18S853/D17S1301 and D9S1122/D10S1435/D12ATA63. The population genetic investigation showed that these 6 loci have high genetic polymorphism, which could be applied in parentage testing or personal identification. These two miniSTR typing systems are valuable in the forensic identification, especially for the highly degraded samples, because of the high sensitivity, species-specificity, tissue identity and genetic stability.