A Multiplex Assay With 55 SNPs For Individual Identification And The Application In Forensic Science

Single nucleotide polymorphism(SNP) is a DNA sequence polymorphism caused by single nucleotide variation. A lower mutation rate comparing with STR makes it an excellent tool for relationship testing and the potential smaller amplicons are required by highly degraded DNA analysis. Otherwise, SNPs could be used as DNA intelligence tools to infer biogeographical ancestry(BGA) and externally visible characteristics(EVCs) that STRs are not especially powerful. As bi-allelic makers, SNPs need to be compensated for with much more markers in order to obtain highly discriminating information.It is valuable to detect dozens of SNPs or more loci simultaneously in forensic science, clinical diagnosis, etiological test and the other subjects. Polymerase chain reaction(PCR) is an elegant technique capable of specifically amplifying a single DNA molecule into billions of copies in a short time. The technique is widely used in molecular biology and the other related fields since 1985, for it is sensitive, specific, productive and time saving. In order to improve the efficiency of the reaction, various aspects should be considered, including the concentration of the components, temperatures and times of each reaction steps, cycling numbers, and so on. It is time consuming and inefficient to adjust each element separately, and the result is sometimes instable. It was even more complicated for multiplex PCR because of the presence of more than one primer pair in the enzymatic reaction.In this research, we took the example of the construction of a 55 Plex SNPs typing assay, to explore the theory of the PCR and discuss the optimization strategy. For a typing method newly established, a series of experiments were performed to assess the forensic application. Part 1 Construction of the 55-SNPs multiplex systemObjective: To develop a stable multiplex system for genotyping 55 SNPs simultaneously.Methods:A multiplex typing assay including 44 SNPs was developed in our laboratories. In this research, it was expanded to a 55 Plex.1 Loci alteration: A loci that didn’t perform well in the expanded system was eliminated, and another 12 SNPs, including a SNP located in Amelogenin for gender identification were added into the multiplex. The newly added SNPs for individual identification were selected from the research of Kidd’s lab.2 Primer design and modification: New primers for PCR amplification and SBE reaction were designed for the 12 SNPs, and the sizes of amplicons were between 56-117 bp. The PCR primers of 4 SNPs that already existed in the original system were redesigned for higher PCR efficiency or lower background peak. Tm and the size of amplicons were considered as priority. A total 28 original SBE primers have been modified, of which 23 were length change and the others were detection orientation alteration.3 Modification of the reaction protocols: The reaction were modified for all the five steps, including several aspects of the method: the total volume, the concentration of the component, annealing temperature and the cycling numbers, etc.First, The total volume of PCR amplifications was reduced from 25μl to 12.5μl, containing 0.5-2ng(extracted by QIAamp Blood midi Kit) or 5-20ng(extracted by Chelex-100) DNA, 1×PCR Gold Buffer, 8m M Mg Cl2, 800 μM of each d NTP, 0.009-0.232μM of each primer and 1 unit of Ampli Taq Gold DNA polymerase. Thermal cycling remain the same as previously.Second, excess of d NTP and PCR primers were then cleaned up by addition of a freshly prepared mix of 1μl(1U/μl) shrimp alkaline phosphatase(SAP) and 0.3μl(10U/μl) Exonuclease â… (Exo â… ) to 2.5μl PCR products and incubated at 37℃ for 1h followed by 15 min at 80℃ for enzyme inactivation.Third, SNa Pshot reaction have also been modified from a volume of 10μl to 4.5μl. SBE primers with the concentration of 0.090-0.903μM were diluted in 160 m M ammonium sulfate to minimize primer-dimer artefacts. SBE reactions were carried out by 2 parallel multiplexes(29Plex and 26Plex), each containing 2μl SNa Pshot ready reaction mix, 1.2μl pured PCR product and 1.3μl pre-mixed SBE primers. The thermal cycling used: 50 cycles of 96℃ for 10 s, 58℃ for 5s, 60℃ for 30 s.Fourth, 1μl SAP was directly added into the SBE product, and then incubated at 37℃ for 1h followed by 15 min at 80℃ for enzyme inactivation.Finally, 1μl of SAP-treated product was mixed with 12μl Hi-Di formamide and 0.2μl of Gene Scan-120 LIZ internal size standard. Separations were made with ABI PRISM 3130 Genetic Analyser.Results: The expanded 55 Plex typing assay was successfully developed, with high efficiency and low cost.Conclusions: A 55-SNPs typing system was developed based on SNa Pshot technique. Here are several approaches for optimization:1 The amplification efficiency of each target in multiplex PCR could be improved by utilization of primers with nearly identical optimum annealing temperatures and shortening lengths of amplicons.2 The amplification efficiencies are high priorities compared with specificities in multiplex PCR, and the latter could be increased by designing SBE primers that don’t overlap with the PCR primers.3 The increase in the number of SBE cycles could increase amount of productions without adverse effects and balance the peak heights between different loci.4 Using SBE primers that are complementary to the opposite strand could possibly reduce the inequality of the two allele peak heights in the same locus. Part 2 Development of the guidelines for allele callingsObjective: To develop specific guidelines for allele callings.1 kits, Bin sets and Panels were set up according to the tutorial. A minimum of 30 RFUs for black and red colours were set as peak thresholds during analysis, respectively. Min Peak Height Ratio was changed to 0.2. Besides the two adjustments above, default values were used for all other fields of the analysis methods.2 The allele calls were labeled automatically with the pre-set panel and analysis method before manually reviewed.3 The options of each electropherogram, including nucleotide lengths and peak heights of all the approved allele calls were exported to Excel, of which the peak height data of heterozygote were separated out to calculate the peak height ratio.Four hundred and ninety samples were typed by the 55 Plex method, the range of the peak heights and the peak height ratios for heterozygotes of each locus were analyzed.Results: Detail guidelines for allele calling were developed.Conclusions: The detailed guidelines were developed based on two parameters: peak sizes and peak height ratios, which were analysed by typing results of fluorescent capillary electrophoresis of 470 samples. It is useful for interpreting results for inexperienced analysts and laboratories and benefits promotion of the method. Part 3 The application study in forensic science for the 55-SNPs multiplexsystem.Objective: To estimate the application value of the 55 Plex typing assay in forensic science.Methods:1 Genetic polymorphism data of 180 samples from unrelated healthy individuals in Hebei Han was investigated with the 55 SNPs and 26 commonly used STRs, population pairwise genetic distance between Hebei Han and the other 176 population groups in Kidd’s research were calculated.2 DNA from 100 father-child-mother trios were typed with the 55 Plex multiplex assay to estimate its value in paternity testing. Methods:3 A series of validation experiments, such as the sensitivity, specificity testing, mixture and degraded DNA studies were performed.Results:1 The minimum allele frequencies were from 0.242 to 0.492, with average 0.390 for the 54 SNPs, which showed a satisfactory polymorphism in Hebei Han population. Pairwise linkage disequilibrium between all the SNPs and the STRs were not observed(P<0.05/3160=0.000016; significant after Bonferroni correction). So “Multiplicative principle” for all the 80 markers could be used when necessary. The combined match probability and cumulative probability of exclusion using the 54 SNPs were 1.327×10-22 and 0.999932 in Hebei Han population, respectively. In the population genetic distance pairwise of each loci with 176 populations, among 5021 comparisons, 3923 showed no significant differences and 1098 showed Fst P<0.05.2 All the conclusion of the paternity testing were consisted with that used by STRs. The combined paternity index(CPI) were from 1.64×103 to 6.39×108,which are equal to relative chance of paternity(RCP) from 0.99939 to 0.9999999984 for 73 non-excluded cases. In the 19000 simulation “father-child-mother trios” paternity testing, all the unrelated males were identified, with 1 to 24 loci for each trio that went against the Mendelian laws.A case was reported that two paternal one-step incompatibilities in both FGA and D10S2325 were observed, as well as a rare allele in D10S2325. The CPI were 4057.5933(less than 10000) using 26 STRs. While all the 54 SNPs were followed the Mendelian laws, and the CPI were 13115.9826. In this case, the 55 Plex assay played a strong supplemental role.3 Complete SNP profile could be obtained from at least 0.125 ng DNA. Species specific study results showed that 15 loci were detected in rhesus monkey samples while no signal was detected in other species. Mixture could be easily identified when two DNA were mixed in seven different ratios(10:1, 5:1, 2:1, 1:1, 1:2, 1:5 and 1:10). The 55 Plex method produced a higher success rate for genotyping with degraded DNA samples compared with Amp F?STR® Identifiler Plus PCR Amplification Kit.Conclusions: All the SNPs in this research showed high polymorphisms and no significant linkage disequilibrium were observed either between any two SNP loci or between SNPs and 26 commonly used STRs in Hebei Han population. The multiplex system have a high effectiveness and good species-specific. Along with the low mutation rates and short amplicons, the 55 Plex typing system is a highly recommended supplementary method in paternity testing and genotyping of degraded samples when STR typing alone fails to apply sufficient information.