Construction Of Four Fluorescence Labeled Multiplex Typing System For Six MiniSTR Loci And Evaluation Of Its Forensic Application

Objective: DNA genotyping for highly degraded samples has been a tough problem in the DNA fields of forensic science. In some murder cases or disasters of major group, forensic scientists had to face a large number of highly degraded samples, which were usually failure to be detected and typed using the commercial multiplex short tandom repeats (STR) kits. As a result, it would delay the case detection and the missing person identification. In recent years, more and more studies had been performed to resolve the highly degraded samples, mainly including two aspects, DNA extraction and DNA typing. For example, the technique of DNA extraction has been improved to acquire DNA fragments as long as possible. On the other hand, some new generation of genetic markers have been used such as single nucleotide polymorphism (SNP) and miniSTR to type short DNA fragments. miniSTR technique was first proposed by Butler, which had been used in the personal identification in the 9.11 case in 2001 and played a very important role in the identification of missing person. miniSTR technology could reduce the size of PCR products by moving the primers as close as possible to the STR repeat region. So miniSTR techonology was applied to examine highly degraded DNA samples and showed higher successful rate than common STR techonolgy. miniSTR has been recommended as a new genetic marker by the European DNA Profiling Group (EDNAP). And recently AB company of USA has already explored a commercial minifiler? kit, including D13S317, D7S820, D2S1338, D21S11, D16S539, D18S51, CSF1PO, FGA, and Amelogenin. The minifiler? kit can solve most of the highly degraded samples. However, these miniSTR loci are not enough to solve some unusual cases. Therfore, in order to increase the power of detection system, National Institute of Standards and Technology (NIST) studied 26 non-CODIS miniSTR loci to examine highly degraded DNA samples. Several of these new miniSTR loci (D10S1248, D14S1434, D22S1045) have been recommended to add in the Europe DNA database by the EDNAP. The United States, Japan, Spain, Singapore, Korean has reported the population genetic data of D1S1677, D4S2364, D10S1248 miniSTR loci and its forensic medicine application value, while our country is lack of relative studies and population genetic data. In the present study, we selected six non-CODIS minSTR loci D10S1248, D2S441, D1S1677, D9S1122, D10S1435, D17S1301 to establish fluorescence-labeled multiplex typing system and investigate the allele frequency in the Han population of Shandong province, as well as evaluated the value of forensic application of the system.Methods:Genome DNA samples were extracted from whole blood of 120 unrelated healthy individuals of Han population of Shandong province by using two-step Chelex-100 method. We constructed two fluorescence labeled multiplex typing system. The first multiplex typing system includes D10S1248, D2S441, D1S1677, and each sense primer of them was labeled with 6-FAM, HEX, TAMRA fluorescence. The second multiplex typing system includes D9S1122, D10S1435, D17S1301, each sense primer of them was labeled with 6-FAM, HEX, TAMRA fluorescence. All DNA samples were multiplex amplified for six miniSTR loci and the amplified products were separated by capillary electrophoresis. The volume of multiplex-PCR was 10μl. The concentration of primer, Mg2+, Taq and the annealing temprature as well as the number of cycle were optimized to get the best PCR condition. The amplified products were analyzed on the ABI PRISM 310 and ABI PRISM 3130 Genetic Analyzer. The fluorescence signals were collected by Date Collection Software 3.1 and the data were analyzed by GeneMapper Analysis Software 3.2. The allele frequencies and genetic polymorphism parameters were calculated to evaluate the genetic polymorphism of the six miniSTR loci. To analyze the genetic stability of the six miniSTR loci, samples of 10 families were examined, which were from parentage testing casewok in the Center of Forensic Medicine Identification of Hebei medical univesity. 9947A DNA sample was diluted to 1ng, 0.5ng, 0.1ng and 0.05ng to test the sensitivity. In addition, the blood was layed outside for 1 week to 8 weeks on summer day to model degraded samples, and the muscle tissue, spleen, skin, kidney from the same corpse placed in 37℃, 100% humidity of the incubator one day to a week to make degraded samples, which were tested by the 6 miniSTR multiplex typing system and the commercial commom STR kit to compare the successful rate of them.Results:1 The construction of two fluorescence-labeled multiplex PCR systems: Two sets of fluorescence-labeled multiplex-PCR typing system for 6 miniSTR loci were established. Each locus was successfully geno-typed in all samples without non-specific amplification product interfering DNA typing. The length of all the amplicon is less than 150bp.2 The results of population genetics: 8, 7, 6, 6, 8, 8 alleles and 20, 18, 12, 17, 20, 22 genotypes were respectively detected in the six miniSTR loci D3S3053, D6S474, D20S482, D1GATA113, D2S1776 and D4S2408. No deviation from Hardy-Weinberg equilibrium was observed in the six loci. The heterozygote observed (Ho) were respectively 0.750, 0.775, 0.650, 0.708, 0.750, 0.858; the polymorphism information component (PIC) were respectively 0.75, 0.71, 0.60, 0.69, 0.73, 0.77; the power of exclusion (PE) were 0.510, 0.553, 0.355, 0.441, 0.51,0.711; and the power of discrimination (PD) were 0.900, 0.881, 0.809, 0.881, 0.893, 0.913 respectively for D10S1248, D2S441, D1S1677, D9S1122, D10S1435 and D17S1301. The cumulative power of exclusion and power of discrimination of the six miniSTR loci were respectively 0.988817 and 0.9999975.3 The results of hereditary stability: Paternity testing of 10 true father-child-mother trios at the 6 loci demonstrated that the parents can transmit their alleles stably to their child, which is consistent with Mendle's law.4 The sensitivity of the two sets of multiplex typing system: The results of the sensitivity analysis demonstrated that 0.05ng 9947A DNA template could be successfully typed by the two sets of fluorescence-labeled multiplex-PCR typing system for 6 miniSTR loci, while the sensitivity of the PowerPlex16 kit was 0.1ng.5 The analysis of degraded samples: For the highly degraded DNA extracted from the tissue of the corpse placed in 37℃, 100% humidity of the incubator for a week and blood layed outside for 8weeks, the mini-STR assays resulted in complete DNA profiling in all of the 6 miniSTR loci, whereas the results of PowerPlex16 kit showed allele dropout or non alleles.6 The study of species-specificity: No specific amplified products were detected in some animals such as monky, pig, cattle,dog, fish, and rabbit.Conclusions: The six miniSTR loci D10S1248, D2S441, D1S1677, D9S1122, D10S1435 and D17S1301 showed high genetic polymorphism and hereditary stability. Two sets of fluorescence labed multiplex-PCR typing system for 6 minSTR loci were established with good sensitivity (0.05ng) and stability, which showed higher successful rate for analyzing the highly degraded samples than the common STR technique. In conclusion, these two minSTR typing system not only are 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.