A Study On The Application Of A Novel Linked Genetic Marker SNP-STR In Unbalanced Mixtures

Background Numerous biological samples in forensic cases originated from more than one source,which brought difficulties to both experimental and explanatory methods applied in DNA mixtures due to the uncertainty about the number of contributors and the mixing ratios.Autosomal short tandem repeats,which had wide distribution and good polymorphisms,had become the targets in daily forensic works.In the last two decades,on the base of STR profiles,the mathematical explanation methods had become more and more objective,e.g.from the likelihood ratio which simply relied on the peak areas and heights to joint Bayesian networks.However,the hypothetical deduction of different theories which obeyed different criteria rose up the suspicion in court.Furthermore,once the mixing ratios surpassed 10:1,the amplification of the minority in the mixture would be inhibited by the mark effect from the major one,which caused the invisible results of the minor contributor in the STR profiles.On the other hand,to enlarge the detectable differences among the contributors,more sensitive biomarkers came out,e.g.Y-STR,single nucleotide polymorphism(SNP),deletioninsertion polymorphism(DIP),which lead the detectable ability for the minority to a new level.However,the limitation in polymorphism blocked the wide usage of these markers in real cases.In recent years,Hall D,Castella V et al.had raised a novel linked genetic marker DIP-STR,which combined the advantages of STRs and DIPs and heightens the mixing ratio up to 1000:1 with normal STR profiling equipment.In 2013,Li W et al.,inspired by the amplification n refractory mutation system(AMRS),succeeded in specifically amplifying the target SNP-STR just in one PCR with three special-designed primers.Therefore,we can explore the application prospects of SNP-STRs in unbalanced mixtures.To quantify the performance of the novel markers and to test them at different mixing ratios,a set of SNP-STRs was needed to be built up.Objective The aims of this study were to build up a multiplex amplification system of SNP-STRs and to detect the feasibility of this system in the unbalanced mixtures.Methods(1)We screened target genes in databases with reference to related documents under the criteria below: 1)The polymorphisms of the STRs must have been tested in the previous reports and all the selected STRs should confirm to linkage equilibrium.2)The SNP located in the up or down stream of the STR sequence,within the distance less than 200 bp.3)The frequencies of the SNPs reported in the database should be more than 0.15 in Chinese Han nationality.4)The probability of informative genotypes of each locus should be more than 0.2451.(2)According to the AMRS,we designed the primers via Pimer 3 for each SNP-STR and then tested the specificity of these primer groups.(3)The construction of the multiplex system was restricted by: 1)The mutual effect within all the primers should be reduced to the least through the adjustment of the consistencies or the sequence design of the primers.2)The peak height of the heterozygotes should be between 1000-3000 RFU when using 2 ng standard,while the homozygote should be between 3000-6000 RFU.(4)We detected the sensitivity of the multiplex system with single-source DNA.(5)The statistical data was collected through 350 unrelated individuals of Han nationality of Hubei province.(6)We simulated the commonest mixtures—two-sources blood mixtures with the DNA samples extracted from blood and the mixing ratios were set in different levels to detect the limit of the multiplex system.Results(1)8 candidates and one Amelogenin gene constituted the multiplex amplification system.(2)All the loci were polymorphic in Hubei Han nationality and the statistic of these loci confirmed to Hardy-Weinberg equilibrium.(3)The sensitivity of the system for the single-source DNA reached 0.05 ng,meanwhile,the max mixing ratio at which the minor contributor can be detected was 20:1.(4)The detectable ratio of part loci can be more than 20:1,and 6 subtypes can even reach 1000:1.Conclusion We successfully built up a SNP-STRs multiplex system and the application of this system in unbalanced mixtures proved feasible.