|
Objective: Since the 80’s in the last century, the short tandem repeats (STR) genetic markers had been generally used in the forensic casework to solve personal identification and parentage testing, because STR are widely distributed in the mankind’s genome and have high genetic polymorphism, as well as easy to detect the genotype. In the present, STR genotype detection has become the main techonology in the forensic science and the STR data become the main composing of DNA database. Using STR typing combined with mitochondrial DNA polymorphism analysis, most of the human personal identification and parentage testing case can be successfully resolved. However, in some forensic casework, the DNA samples were highly degraded due to exposure to environmental elements or natural contaminants. These highly degraded samples were usually failure to type using the commercial multiplex STR kits, which showed either loss of information at higher molecular weight allel or loss of signal. This problem became more obviously with the increase of the PCR products during PCR. 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. Several of these new miniSTR loci have been recommended to add in the Europe DNA database by the European DNA Profiling Group (EDNAP). The United States, Japan, Spain, Singapore, Korean has reported the population genetic data of some miniSTR loci and its forensic medicine application value, while our country is lack of relative study and population genetic data. In the present study, we selected D3S3053, D6S474, D20S482, D1GATA113, D2S1776 and D4S2408 six miniSTR loci to investigate the genetic polymorphism in Han population of Hebei province China and evaluated its application in forensic science.Methods:Genome DNA samples were extracted from whole blood of 120 unrelated healthy individuals of the Hebei province Han population by using TIANGEN blood genome DNA extraction kit. Forward primers of D20S482 and D2S1776 were fluorescently labeled by 6-FAM in 5’end, D3S3053 and D1GATA113 labeled by HEX in 5’end, D6S474 and D4S2408 labeled by TAMRA in 5’end. All DNA samples were multiplex amplified for six miniSTR loci. D3S3053, D6S474 and D20S482 were multiplex amplified; D1GATA113, D2S1776 and D4S2408 were multiplex amplified in 10μl reaction volume with specific primer sets. After 28 cycles, the multiplex-PCR products were analyzed on the ABI PRISM 310 Genetic Analyzer. The fluorescence signals were collected by Date Collection Software 3.1 and the genotype were analyzed by GeneMapper Analysis Software 3.2. The allele frequencies and genetic polymorphism parameters were caculated to evaluate the genetic polymorphism of the five miniSTR loci. To analyze the genetic stability of the six miniSTR loci, samples of 9 families were examined, which were from parentage testing casewok in the Center of Forensic Medicine Identification. The DNA were extracted from the heart, liver, spleen, lung, kidney, brain tissue, muscular tissue, skin and blood stain from the same corpse to test the tissue identity. 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, which were tested by the 5 miniSTR multiplex typing system and the commercial commom STR kit to compare the successful rate of them.Results: 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. 6, 6, 7, 8, 8, 7 alleles and 14, 16, 15, 27, 27, 23 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 0.652,0.758,0.758,0.667,0.617,0.85; and polymorphism information component (PIC) were respectively 0.64,0.68,0.69,0.83,0.85,0.83 for D3S3053, D6S474, D20S482, D1GATA113, D2S1776 and D4S2408. The power of exclusion (PE) were 0.298,0.523,0.523,0.313,0.271,0.662;and the power of discrimination (PD) were 0.862,0.857,0.877,0.943,0.947,0.917respectively for D3S3053, D6S474, D20S482, D1GATA113, D2S1776 and D4S2408. The cumulative power of exclusion and power of discrimination of the six miniSTR loci were respectively 0.97 and 0.9999994. Family analysis of 27 individuals from 9 families at the 6 loci demonstrated that the parents can transmit their alleles stably to their child, which is consistent with Mendle’s law. The genotypes of the different tissues in the same cadaver were identical, indicating the tissue identity of the 6 loci. 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 IdentifilerTM kit was 0.1ng. For the highly degraded DNA extracted from blood layed 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: The six miniSTR loci D3S3053, D6S474, D20S482, D1GATA113, D2S1776 and D4S2408 showed high genetic polymorphism, tissue identity 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.
|