Investigation Of The Genetic Polymorphism Of The Six MiniSTR Loci In Hunan Han Populations

Objective: Short tandem repeat (STR) genetic marker system, which is widely distributed in human genome and has high genetic polymorphism and the fast test means, has became the mainstream technology in forensic identification practice. However, in many other forensic cases, DNA samples are highly degraded for many reasons, a loss of signal is typically observed with larger-sized STR products and it is difficult to have a complete genotype when using current commercially conventional multiplex STR kits. 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 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. Many studies about miniSTR,miniX-STR and miniY-STR have shown its value in the fields of forensic science. When applied to forensic science, accurate, rapid and convenient typing detection is crucial. To confirm the accuracy of DNA typing, constructing a standard allelic ladder (AL) is important. Only when we have a set of precise allelic ladder system, which was named by international standard, it would be possible to make a right typing and change data between different labs in the world. Therefore, to explore more miniSTR typing systerms to be used in the detection of highly degraded samples, in the present study, we constructed the standard AL for D10S1248, D2S441, D1S1677, D9S1122, D10S1435 and D17S1301 six miniSTR loci by molecular cloning technology, and investigate the genetic polymorphism of the six miniSTR loci in the Han population in Hunan Province and evaluated the value of constructing allelic ladder in the court practice.Methods: Genome DNA samples were extracted from whole blood of 155 unrelated healthy individuals of the Hunan Han population by using chelex 100. After being amplified by PCR, different miniSTR allelic fragments were isolated from the 10% denaturing PAGE electrophoresis and sliver staining. The miniSTR allelic fragments were cut from the gel and recycled by Wizard SV Gel and PCR clean up system and then were blunt-end subcloned individually into the pGEM-T plasmid vectors. Next the pGEM-T plasmid vectors were transfected into competent E.coli DH5αcells, which were cultivated on selective media. Positive clones were selected by 4% agarose gel electrophoresis. Then the size and structure of the inserts were confirmed by DNA sequencing. Each allele was named according to the principle recommended by ISFH. The recombinant plasmids DNA with the inserts were then used as template for reamplification. Adjusting the proportion of each component in accordance with band intensity or quantitative results, the peak height of all alleles was made to be approximately equal. Then the PCR products of all alleles were mixed to generate the standard allelic ladder for each locus. Using these AL, the genetic polymorphisms of six miniSTR loci in Hunan Han population were studied. Forward primers of D10S1248 and D17S1301 were fluorescently labeled by 6-FAM in 5'end, D2S441 and D9S1122 labeled by HEX in 5'end, D1S1677 and D10S1435 labeled by TAMRA in 5'end. They were amplified then both the products and the allelic ladder were analyzed on the ABI PRISM 310 Genetic Analyzer. 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: Standard allelic ladder of six miniSTR were constructed by technology of molecular cloning. 9, 7, 7, 6, 7 and 8 alleles were respectively detected in the six miniSTR loci D10S1248, D2S441, D1S1677, D9S1122, D10S1435 and D17S1301.The sequencing results confirmed that the size and the repeat unit of the six loci were respectively [GGAA], [TCTA], [GGAA], [TAGA], [TATC] and [AGAT]. Investigation of population genetics showed that 9, 7, 7, 6, 7 and 8 alleles and 21, 18, 15, 15, 22 and 20 genotypes were respectively detected in 155 unrelated healthy individuals. No deviation from Hardy-Weinberg equilibrium was observed in the six loci. The heterozygote observed (Ho) were respectively 0.729, 0.735, 0.665, 0.787, 0.677 and 0.671; and polymorphism information component (PIC) were respectively 0.699, 0.716, 0.591, 0.698, 0.752 and 0.663 for D10S1248, D2S441, D1S1677, D9S1122, D10S1435 and D17S1301. The power of exclusion (PE) were 0.475, 0.735, 0.376, 0.575, 0.384 and 0.385;and the power of discrimination (DP) were 0.888, 0.889, 0.814, 0.875, 0.921and 0.869 respectively. The accumulated power of exclusion and power of discrimination of the six miniSTR loci were respectively 0.98 and 0.999997.Conclusions: Using molecular cloning technology, we constructed six sets of allelic ladder for six miniSTR loci including D10S1248, D2S441, D1S1677, D9S1122, D10S1435 and D17S1301, each contains 9, 7, 7, 6, 7 and 8 alleles respectively. These sets of allelic ladder are the key component of miniSTR typing systerm. The investigation of population genetics using these ladders showed that the six miniSTR loci have high genetic polymorphism, which could be used for personal indentification and paternity testing in forensic medicine, especially be valuable for the DNA typing of highly degraded samples.