DIP-STR Makers For The Analysis Of Unbalanced DNA Mixtures

Background Since the advent of DNA typing in the 1980 s,significant changes have taken place in the field of forensic science,as an important part of criminal science and technology,forensic DNA technology is playing an increasingly important role in combating crime.DNA technology can be used to identify various biological materials extracted from the crime scene,including hair,semen,bone,cigarette butts,clothing and other on-site physical evidence(stains or tissues).With the development of forensic DNA technology,trace DNA or degraded DNA samples can also be better identificated.Mixed stains are common biological physical evidence with non-single source and uncertain components.Particularly,there are a large number of highly unbalanced mixed DNA samples,bring great difficulties to the identification work in routine examination,resulting in failure to effectively support the case investigation.These bring great difficulty to the inspection and verification work,resulting in the inability to effectively support the case investigation.Conventional detection method for modern forensic laboratory is based on STR(short tandem repeats)site,with good polymorphism widely distributed on autosomal DNA.However,this method usually cannot accurately read the typing results of secondary donor DNA when the ratio of mixed DNA samples exceeds 10: 1.For example,in sexual assault and violent crimes,the ratio of major and minor contributor's DNA is unbalanced,and it's difficult to obtain the STR typing result of minor donor in mixed DNA by the conventional STR detection method.The major reason for these phenomena is that the unbalanced ratio of the DNA of the victim 's DNA to the suspect 's DNA in these mixture,which leads to the masking effect of PCR amplification.The signal of the major DNA sample interferes with the minor DNA sample,making it impossible for the trace DNA samples in the mixed sample to be fully amplified and detected so as to get the typing.As a result,the issuance of appraisal results and the investigation of the case cannot be affected.Recent studies have shown that the novel linkage genetic marker DIP-STR,which refers to the genetic marker of the DIP(deletion insertion polymorphisms)polymorphism fragment and the standard short tandem repeat sequence-STR,can target trace DNA in mixed DNA samples,using inter locking allele-specific primers to target trace amounts of DNA in mixed DNA samples.Due to the sensitivity of DIP and the polymorphism of STRin the linkage markers,when the mixing ratio reached 1:1000,the sensitivity and specificity of DIP-STR markers remained ideal.This method not only breaks the amplification masking effect,but also is not limited by the sex of the DNA donor in mixed stains.Therefore,this technique isconsidered to have great potential in solving the problem of DNA mixtures.However,the number of DIP-STR genetic markers reported at home and abroad is limited,which has greatly limited its discrimination ability and practical application.To solve this problem,it is urgent to explore more DIP-STR loci and construct DIP-STR marker groups,so as to utilize enough loci to achieve higher discrimination ability.Objective Screening and verifying the new DIP-STR genetic markers,optimizing the chain marker amplification system,constructing an integrated technology platform,and further exploring the potential application of such genetic markers in unbalanced mixed DNA samples.Methods(1)The screening criteria were established to pre-screen the target sites,combining with the research methods reported in the literature,as well as referring to the database and literature.(2)According to the pre-screened sites,Primer 3 software was utilized to design specific primers for target sites,and the amplification system was constructed.The DNA amplification products were sequenced and compared to optimize the corresponding primers,amplification system,and further screening sites..(3)A total of 200 individuals from Chinese population were randomly collected for polymorphism test of secondary screening loci and the corresponding random matching probability of DIP-STR allele frequency and DIPSTR genotype was calculated using Powerstats V12(Promega)software.The parameters of Panel and bins for DIP-STR were designed by combining the results of population data and sequencing to realize automatic analysis.(4)The detection thresholds of each single marker were explored by using single DNA sample with different concentration gradient.(5)The mixed DNA samples of two individuals with different ratio were simulated and the sensitivity of DIPSTR genetic marker was detected.Results(1)Among the 5 pre-screened DIP-STR genetic markers,three of them had a relatively high probability of informative markers(I value),which indicates that it has potential application prospect in individual recognition.(2)The genetic polymorphism and power of discrimination of the above three sites are different,which can be further tested in practical case tests.(3)The detection threshold value of single-source DNA samples can be as low as0.005 ng.(4)The comparison between DIP-STR and autosomal STR markers shows that when the ratio of components increases,the autosomal STR markers are difficult to genotype the minor DNA due to the interference of a large number of major DNA,while the DIP-STR genetic markers show some superiority and can detect the minor DNA at the ratio of 1:1000.Conclusion Through screening,three DIP-STR markers with good sensitivity and specificity were obtained in this study,and they also show good polymorphism in a certainpopulation.They have certain application value in the testing of mixed DNA samples,especially mixed DNA samples with unbalanced proportions.