Construction Of Four Fluorescence Labeled Multiplex Typing System For Four X-STR Loci And Population Genetic Investigation

Objective: X-STRs is the short tandom repeats (STRs) on X chromosome. Due to the characters of their high stability, high polymorphism and unique mode of inheritance, X-STRs have attracted much more attention of forensic scientists for analyzing complex kinship cases. Although the existence of X-STRs, i.e. HPRTB, ARA and DXS981 were reported rather early, the amount of X-STRs which have obtained population genetic data and are available for forensic applications is only more than 30. Lack of population data, ethnic difference, linkage and linkage disequilibrium between markers all affect applications of X-STRs for forensic medicine, Therefore, it is particularly important to find more available X-STRs.The purpose of this project is to establish the fluorescent multiplex X-STRs system of 4 unreported X-STR loci, namely GATA151A05 /GATA2B05/DXS7129/HUMUT1595, and to investigate the genetic polymorphism of 4 X-STRs in Han population of the Northern China to provide population genetic data.Methods:1 Loci selection: Four unreported X-STR loci were selected form NCBI GENE database. The four loci located in the same linkage group, and the physical distance between each two loci was less than 10Mb.2 Construction of multiplex typing system: DNA was multiplex amplified for 4 selected X-STR loci. Forward primers of GATA2B05, HUMUT1595 were fluorescently labeled by 6-FAM in 5'end, GATA151A05 by HEX, DXS7129 by ROX. The multiplex amplification conditions such as the concentration of primer, Mg2+, Taq enzyme, anneal temperature, cycle times were optimized. The PCR products were separated electrophoretically on ABI 310/3130 Genetic Analyzer, and fragment size and genotype were analyzed using GeneMapper3.2 software. Each kind of alleles of 3 X-STR loci is randomly selected and sequenced from male samples. The alleles in each locus were named according to the sequencing results and the principles of the international society of forensic haemogenetics (ISFG).3 Population genetic investigation and forensic application: Population genetic investigations were performed on 214 healthy unrelated individuals of Han population in Northern china (105 males and 109 females) using this multiplex PCR system. The results were analyzed by Arlequin3.11 software, and forensic genetic parameters were calculated using online tools (http://www.chrx-str.org). Ten affirmed single-parent kinship test (4 father/daughter duos and 6 mother/son duos) were analyzed using this 4 X-STRs multiplex system. The tissue identity tests were performed for the 4 X-STRs multiplex systems.Results:1 Development of the multiplex PCR system: the fluorescense multiplex PCR system of GATA151A05/GATA2B05/DXS7129/HUMUT1595 was successfully established. Multiplex PCR was performed in 20-μl reaction volumes containing 6pmol of GATA151A05, 5pmol of GATA2B05, 4 pmol of DXS7129, 5pmol of HUMUT1595 primers, 1 unit of Taq DNA polymerase, 1.5mM MgCl2, using Hot start PCR to amplification. A balanced amplification were performed between each of the loci and alleles. This multiplex PCR system was of high genetic stability and good repeatability.2 Results of population genetic investigation: According to the population genetic investigation results, among the 109 cases of female samples, 5 alleles and 8 genotypes were detected in the DXS7129 locus; 5 alleles and 9 genotypes were detected in the GATA151A05 locus; 8 alleles and 14 genotypes were detected in the HUMUT1595 locus; 8 alleles and 18 genotypes were detected in the GATA2B05locus. Among the 105 cases of male samples, 5 alleles in the DXS7129 locus; 6 alleles were detected in the GATA151A05 locus; 7 alleles were detected in the HUMUT1595 locus; 8 alleles were detected in the GATA2B05 locus. Besides GATA2B05 locus, No deviation from Hardy-Weinberg equilibrium was observed in other three loci in the female samples (P>0.05). Each kind of alleles of 3 X-STR loci is sequenced and forensic genetic parameters were calculated base on the population genetic data of 3 X-STR loci. No population differentiation between female and male frequencies, allele frequencies from female and male sample are pooled in 3 X-STR loci(0.00309—0.7678). Linkage disequilibrium was tested, separately, in both female and male samples and association was not detected between alleles in any of the pairs of the 3 X-STR loci. The heterozygote observed (Ho) and polymorphism information component (PIC) in DXS7129, HUMUT1595, GATA151A05 locus were respectively 0.29358, 0.60550, 0.54128 and 0.36858, 0.62258, 0.58478. The power of discrimination for females (PDfemale) and the power of discrimination for males (PDmale) were respectively 0.593145, 0.761857, 0.731926 and 0.385692, 0.602436, 0.573134. The mean exclusion chance (MEC) for ChrX markers in trios involving daughters and MEC for ChrX markers in father/daugther dous were respectively 0.356211, 0.522350, 0.487274 and 0.226958, 0.380390, 0.346576.3 Pedigree investigation: No mutation was found in the ten affirmed single-parent kinship test (4 father/daughter duos and 6 mother/son duos). The results verified the unique genetic mode of X-STRs.4 Tissue identity test: The genotypes of the different tissues in the same cadaver were identical, indicating the tissue identity of the 4 X-STRs loci. Conclusions: A four fluorescense multiplex PCR system of GATA151A05/GATA2B05/DXS7129/HUMUT1595 was successfully constructed with accurate genotyping results and good repeatability. The population genetic investigation showed that HUMUT1595 locus was of high polymorphism, and GATA151A05 locus was of medium polymorphism. Both of them are available for forensic kinship test and personal identification. This study enriched the X-STRs population genetic data.