The Genetic Polymorphism Of18STR Loci And Its Application For Forensic Science

Background and significanceShort tandem repeats (short tandem repeat, STR), are also called microsatellite DNA (microsatellite deoxyribonucleic acid) or simple sequence repeat (simple sequence repeats, SSRs). The STR core sequence is generally constituted by2to6nucleotides, because the core sequence number series was repeatedly arranged differently in different individuals, it showes length polymorphism. Fragment length is short, so it has high amplification efficiency, even serious degraded or decay samples or long time placed samples can be successful genotyped, forensic DNA laboratory in the world are using this technology for individual identification and kinship analysis and identification. In1986, China began to use forensic DNA analysis techniques in applied research, and it was applied to the actual prosecution case in1989. Detection means included RFLP analysis to PCR-silver staining banding typing, played an important role, but forensic experiment prosecution case requires a big difference to the present. Till now, fluorescently labeled STR multiplex amplification greatly expand the forensic DNA testing technology applications range and has become a major means of inspection.For DNA Amplification Kit, China’s public security office with DNA laboratory rely on imported commercial kit in the daily case in the absence of a localization kit, these commercial kit clearly has a lot of advantages:the results are stable, reliable, good repeatability, but it also has its drawbacks:expensive and part of the loci it contains have low polymorphism in the Chinese population. Evidence Identification Center of the Ministry of Public Security presided over the research and development of DNATyperTM15Amplification Kit, with four-color fluorescence detection of15loci amplified simultaneously (including gender sites) in the "10th Five-Year Plan" period. However, with the continuous development and a wide range of applications of the technologies of DNA technology, the kit of the15loci already can not satisfy the needs of the subject case, mainly amount information of amplification can not satisfy the ratio of the information on the requirements of the mass DNA data, and compared with similar foreign products, such as Promega Corporation PowerPlex@21amplification kit (including gender sites), the gap is significant, the latter increased amount of information detected and saved samples and time which improve efficiency. Therefore, the Ministry of Public Security Evidence Identification Center recently developed a DNATyperTM15Plus Amplification Kit, which can amplify18loci. Our study is using the kit to amplify a large number of Han and Uyghur population and get their allele frequencies and forensic application parameters which provide the basic data for the calculation of the detection system of individual identification and paternity identification. The scientific basis for other DNA laboratory to select an appropriate kit is provided through measuring the test performance of the kit on biological samples on-site inspection.Research MethodsThe samples used in the study:1.1500Guangdong Han unrelated individuals blood sample (filter paper) are from the DNA laboratory of Guangzhou forensic institute.2.1381Xinjiang Uygur unrelated individual blood samples (filter paper) are from Xinjiang kash Public Security office collected by informed volunteers. 3.2800M Control DNA is taken from Promega Corporation, the concentration is10ng/μl.4. The admissibility of evidence from Panyu District, Guangzhou City Public Security Bureau Branch of the DNA laboratory, after successfully amplified by AmpFSTR(?) SinofilerTM Amplification Kit, we selectd210Amplification product,60of them were buccal swabs,30blood on the scene,30live cigarette butts and the30hard tissue (15nails,15cartilage),30mixed semen stains (underwear and vaginal swabs),30contact samples (including10glass wipe,10palmprint and10clothing). This study use DNATyperTM15Plus amplification kit to test the1500unrelated individuals in Guangdong Han and1381unrelated individuals of Uygur from Kashi Xinjiang. U.S. AB9700PCR thermal cycler is used for amplification, and3130xl automatic genetic Analyzer is used to detect it. We analyze electrophoresis results by GeneMapperlD v3.2. According to the results of genotyping18STR loci, we use powermarker3.25software to calculate gene frequencies, and the distribution of gene frequencies is conducted by chi-square test, forensic statistical software Powerstats1.2is used for the Calculation of paternity exclusion (Excluding probability of paternity PE), matching probability (Probability of Matching Pm), discrimination power (probability of discrimination DP), polymorphism information content (polymorphism information Contents PIC), and calculate the cumulative individual identification rate (cumulative probability of discrimination CDP) of the18STR loci, the cumulative probability of paternity exclusion (cumulative probability of exclusion CEP). All of that provide basic information for individual identification, paternity testing and population genetics.210of selected samples amplicated successfully by AmpFSTR(?)amplification SinofilerTM were chosen and amplificated by DNATyperTM15Plus amplification kit under the same conditions and in the above cases, from the sensitivity, the detection rate of accuracy, the average peak to compare these aspects of the high and the average peak area.Result1、In1500Guangdong Han unrelated individuals,18STR loci were found 232alleles. Loci FGA had the most alleles,23, while TPOX had the least, only7.18STR loci had single gene frequency in the range of0.0003to0.5407, which was the highest frequency of alleles for TPOX alleles8(0.5407).18STR-loci’s heterozygosity (heterozygosity H) was between0.5920and0.9073, and individual ability (DP value) is the range of0.790to0.984, polymorphic information content (PIC) value in the range of0.5501to0.9010; for paternity exclusion rate (PE value),the most was PentaE(0.810), minimum TPOX(0.281); loci coupling probability was1.33275E-22; cumulative discrimination power was greater than99.99999,999,999,999,99999%, the cumulative probability of paternity exclusion was approximately0.999999964.2、In1381Xinjiang Uygur unrelated individuals,18STR loci were found231alleles, for loci Penta E,it had the most allele (22), why for TH01,the least allele, only six.18STR loci had allele frequencies in the range of0.0004to0.5304, TPOX had the highest frequency of allele8(0.5304)18STR loci heterozygosity (H value) ranged from0.6358to0.9233. individual ability(DP values) were between0.880to0.988, polymorphic information content (PIC) value were in the range of0.5870to0.9180;18loci coupling probability was2.3764E-23, cumulative individual identification ability is greater than99.99999999999999999999%, the cumulative probability of paternity exclusion is greater than0.9999999.3、After the comparation between the amplification kit DNATyperTM15plus and AmpFSTR(?)SinofilerTM Amplification Kit on common samples on-site inspection, in terms of sensitivity, detection rate, accuracy, allele peak height and peak area. DNATyperTM15plus Amplification Kit can test the amount of DNA template of the Control DNA2800M in between0.313ng～1.25ng and get good genotyping map; AmpFSTR(?)SinofilerTM Amplification Kit can test amount of DNA template of the2800MControl in between0.156ng to1.25ng and successfully get a good genotyping map. For210samples, by using the DNATyperTM15plus amplification kit we detected a total of187cases, the detection rate was89.05%.while for using AmpFSTR(?)SinofilerTM amplification kit,100%detected. The amplification kit AmpFSTR(?)SinofilerTM amplified can detect six cases of mixed plaques, and DNATyperTM15plus amplification Kit amplification detected only two cases. Using two kits for the same sample, in the same allele can100%get the same rate of STR typing. Six groups of samples at18STR loci and gender loci, alleles average peak height difference of (AmpFSTR(?)SinofilerTMmplification Kit/DNATyperTM15plusamplification kit) were between0.97-48.05times. The average the peak area difference(AmpFSTR(?)SinofilerTM amplification Kit/DNATyperTM15plus amplification kit difference was an average peakarea) between times0.42times and46.33times and consistent with the value of average peak height, which confirmed the accuracy of the results.Conclusion1.18STR loci of DNATyperTM15plus have highly polymorphism in the Han population and the Uighur population and meet the needs of the the groups individual identification and paternity testing. There is significant difference between two populations’genotyping, indicating that in addition to forensic applications, the detection system also have important applications in archeology, population genetics and anthropology research.2. DNATyperTM15plus kit was used to test210common samples which had been successful tested by Sinofiler Amplification Kit test, we found DNATyperTM15plus kit can accuratly genotype daily common samples. The samples with amount of template between0.313ng-1.25ng can get a good map.3. DNATyperTM15plus kit includes18loci, identified for the single parent and the nameless corpse more favorable inspection as a domestic kit, the price advantage is obvious. It can100%test the blood samples for building a database, the price advantage is obvious, and it meets the demand to build a criminal recordlibrary. For common samples,the detection rate of DNATyperTM15plus kit is less in Sinofiler kit, it is suggested totest fresh samples and a large amount of samples can select DNATyperTM15plus kit.