The Construction And Application Of A Multiplex Typing System With 44 SNP Loci

Objective: The current commercially multiplex STR kits used in forensic DNA typing can solve most cases of individual identification and parentage testing. However, in many other forensic cases that DNA samples are highly degraded due to exposure to environmental elements or incorporation of natural contaminants, STR analysis becomes impossible and it makes the case investigations difficult. The analysis of mitochondrial DNA (mtDNA) hypervariable regions is typically used, but given the haploid and maternal-only transmission of mtDNA, and the power of discrimination is low, its utilization is limited. MiniSTR assays, which is to reduce the size of the PCR products by moving primers as close as possible to the repeat region, produces a higher success rate for genetyping with degraded DNA samples. However, while bringing the primers of STRs closer to the repeat region reduces overall amplicon sizes, the production lengths of loci with the largest range of alleles will be still not less than 100bp. Another drawback to miniSTR approach is that more than one multiplex amplification are required in order to match the discrimination power, but an examiner may not have the luxury of obtaining an abundance of degraded DNA from a limited forensic sample. Single nucleotide polymorphisms (SNPs) are a type of polymorphism involving variation of one single base pair that occurs at appreciable genetic sequence in human genome. SNPs has low mutation rates and can be genotyped with high-throughput technologies and analysed after PCR amplification of very short DNA regions, thus makes them ideal markers for human identification.With a reliable multiplex procedure, many SNPs can potentially be typed using very short recognition sequences—in the range of 45~55 bp (merely the length of the two flanking PCR primers). Otherwise, there are various SNP multiplex typing platforms that are available for simultaneous multiplex detection of 12 or more SNPs, such as the commercial reagent kit SNaPshot which bases on the principle of single base extention technic. Thus, SNPs analysis will clearly be extremely valuable when DNA samples are severely limited or degraded. However , for individual identification SNPs requires high heterozygosity and low coefficient of inbreeding, and should be genotyped with high-throughput technologies, there is not a final panel of individual identification SNPs that had been defined yet. Thus, the objective of this study is to select a sufficient number of autosomal SNP loci that meet the criteria of individual identification (individual identification SNPs,IISNPs) and match the discriminatory power, then develop a highly efficient DNA typing strategy for analyzing all loci simultaneously by using multiplex PCR in association with fluorescent labelled single base extension (SBE) technique, and perform a series experiments to assess the forensic application of the high-throughput SNP-typing methods.Methods:①At least 50 autosomal SNP loci that met the criteria of individual identification were selected. Primers for PCR amplifying and single base extention were designed using primer designing software according to the reference sequence from dbSNP (http://www.ncbi. nlm.nih.gov/SNP).②The multiplex PCR system with all SNPs was developed, then the PCR product was divided into two SBE multiplexes after purified by Exonuclease I and shrimp alkaline phosphatase. The SBE reaction was performed by SNaPshot kit and the excess ddNTPs were removed by addition of shrimp alkaline phosphatase to the SBE mix. The SBE product was detected by ABI 3130 Genetic analyzer, and the data was collected by 3130 Data Collection V3.0 software, fragment size determination and genotyping of all the SNPs loci were done with Genemapper ID V3.2 simultaneously. The amplification conditions such as the concentration of Mg2+, dNTP and primers, the anneal temperature and the cycle times were optimized to balance the PCR product of each locus, so that the final multiplex typing system with all SNPs was developed.③Genetic polymorphism data of 79 samples from unrelated healthy individuals in Hebei was investigated with the SNPs multiplex typing system. A series of experiments were performed to validate the characteristic of the SNPs multiplex typing system, such as sensitivity, species specificity and concordance of different tissue from a same body. Performance of the SNPs multiplex typing system in paternity testing and highly degraded samples was assessed in same cases.Results:①50 autosomal SNP loci were selected and the primers for PCR amplifying and single base extention were designed. All multiplex PCR primers were selected to have theoretical melting temperatures of 60±5℃and the lengths of the products ranged from 69 to 125bp. The lengths of the SBE primers were between 20 and 83 nucleotides, with size intervals of 3 to 6 nucleotides, which were increased by 5'end addition of poly-T or poly-CT.②6 SNPs were eliminated because of instability typing results,then the multiplex typing system was successfully developed with 44 SNPs remained after optimized. All the amplicons with 44 SNP loci could be simultaneously amplified in a single tube and were assigned into two SBE reactions, with 26 loci in Plex 1 and 18 loci in Plex 2 respectively.③The population genetics data of 79 unrelated individuals for the 44 SNP loci was collected. The genotypes of all SNP loci studied were distributed as expected based on the assumption of Hardy–Weinberg equilibrium. The average match probability is 5.59×10-19, and the cumulative exclusion power is 0.9992633421. A complete SNP profile was obtained for 9948 standard DNA from only 0.0625ng. Species specific study results showed that 16 loci were detected in rhesus monkey samples while no signal was detected in other species. Conclusions from the 44 SNPs multiplex typing system in paternity testing were concordance with that from AmpFlSTR? Identifiler? kit. While the 44 SNPs multiplex typing system produced a higher success rate for genetyping with degraded DNA samples compared with AmpFlSTR? Identifiler? kit and miniSTR typing assay, it would be more valuable in analyzing samples which loosed signals by STR typing.Conclusions: By the way of designing PCR and SBE primers,we successfully developed a SNPs multiplex typing system, which including 44 SNPs with high heterozygosity and low coefficient of inbreeding. The application study indicated that the 44 SNPs multiplex typing system could be applied in forensic routine work, and it would serve an important role for analyzing challenging forensic samples.