LDR-based Methods For SNPs Typing Of Degraded DNA And Trace DNA

Background and objectivesFor the past decades,the use of DNA analysis must be the most significant reformation in forensic fields.However,DNA degradation and trace DNA have always been special challenges for forensic scientists obtaining reliable and sufficient genetic information for personal identification and parentage testing.To improve and assess the performance of different genotyping technologies in those cases where DNA is degraded,it would be desirable to have a reference sample of degraded DNA with known fragment sizes.Several methods that may obtain a source of artificially degraded DNA,including ultrasonication,DNase digestion and other physical methods(temperature and humidity,etc) have been reported so far,but there has not been a method accepted extensively.Therefore,we proposed to develop a method to prepare artificially degraded DNA under controlled conditions.For the past few years,two new genetic markers have been developed,i.g. mini-STR and single nucleotide polymorphism(SNP).For forensic analysis of degraded DNA,it has become the essential strategy to reduce the amplicon length as short as possible.The typical technologies are mini-STR-PCR and mini-sequencing. However,the DNA fragment size should be more than 100～200bp when these two assays are used.If the average DNA fragment length is less than 100 bp,how can we get sufficient,reliable genetic information for forensic DNA analysis? It is still a big problem unsolved.Nowadays,two main strategies are used in forensic DNA analysis of low-copy-number(LCN) samples:(1) enhancing STR-PCR assay.By increasing PCR cycle number,this method can give improved results.(2) nest PCR or whole genome amplification(WGA) combined with STR-PCR.After the use of WGA or nest PCR, the quantity of the starting DNA template increases greatly so that subsequent STR-PCR assay does not need much more cycle number.Because LCN samples only contain trace amounts of the starting DNA template,the stochastic effects will increase greatly,and result in allele imbalance and stutter product formation.In addition,it is often impossible to examine the LCN samples repetitively and difficult to assess its reliability.Therefore,the resulting profiles of both strategies above may be complex and difficult to interpret for forensic applications.For the past decades,SNPs typing technologies based on ligase detection reaction (LDR) have developed quickly,and propose a new chance for forensic analysis of degraded DNA and trace DNA.First of all,LDR probes only need short target-specific sequences to hybridize to DNA template.The length of target-specific sequences should(1) be able to recognize the target locus to be tested,and(2) be suitable for recognition and binding by DNA ligase.Therefore,LDR probes can be as short as several dozens of nucleotides.In addition,LDR possesses high fidelity and stringency for base matching at the nick.Theoretically,LDR can be used for SNPs typing as long as the DNA fragment size is not less than 30～50bp.Thus,LDR is suitable for SNPs typing of highly degraded DNA.After recognizing and hybridizing to target loci,padlock probes can be transformed circular molecules sealed by covalent bond.Reliable allelic discrimination achieves because of high fidelity and stringency of LDR.Subsequently,circularized padlock probes can be amplified for signal enhancement by hyper-branched circle amplification(HRCA).The amplification efficacy of HRCA is so exceeding that a great quantity of products can be obtained from trace DNA template.Therefore, LDR-HRCA may be valuable for SNPs typing of LCN DNA.MethodsTwelve human peripheral blood samples(male 6,female 6) were provided by department of forensic Serology,forensic college,China Medical University.Genomic DNA was extracted by the standard phenol/chloroform procedure after digestion with proteinase K.During the treatment of genomic DNA by ultrasonication or DNase digestion, aliquots were taken at a certain time-point,and the DNA was electrophoresed in agarose gel and amplified with the primers designed by ourselves.Therefore,the sizes of DNA fragments were clearly determined.After preparation of the artificially degraded DNA,multiplex STRs loci were typed with AmpFl STR(?) Identifiler(?) PCR Amplification Kit.At the second step,four SNPs loci were selected(rs17750303,rs2307647,rs2307557,and rs17250992),and their LDR probes were designed.LDR probes were used to discriminate alleles by thermostable ligation reaction.Then,PCR was used to amplify the ligated LDR probes for signal enhancement.The genotypes of each locus were identified according to results of gel electrophoresis.A series of LCN samples were prepared by diluting the starting DNA(500,100, 50,40,30,20,and 10 pg/μl,respectively).At the first step,AmpFl STR(?) Identifiler(?) PCR Amplification Kit was used to test the LCN samples.At the second step, Rs17750303 locus was selected,and allelic padlock probes(CPP and APP, corresponding to C-allele and A-allele respectively) were designed.Padlock probes were used to discriminate alleles by thermostable ligation reaction.Then,HRCA was used to amplify the circulized probes for signal enhancement.The genotypes of each locus were identified according to results of gel electrophoresis.Results1,After genomic DNA was ultrasonicated,the average sizes of DNA fragments varied significantly.The repeatability of ultrasonication was poor.As the digesting time extended,the average sizes of DNA fragments reduced gradually.After 1μg genomic DNA was digested with DNaseⅠfor 150 min,the fragment sizes of degraded DNA were not more than 100 bp.2,Using AmpFl STR(?) Identifiler(?) PCR Amplification Kit,no STR alleles could be obtained from the degraded DNA with fragment sizes less than 100bp.Using our LDR-PCR method,clear allelic discrimination of each target locus was achieved after staining of the final reaction mixtures with EB and visualization by UV illumination. The types of base variants did not influence the fidelity of DNA ligase.The target-specific sequence of LDR probes could be as short as 11 nucleotides,which did not disturb the ligation reaction.2 or 4 SNP loci could be correctly discriminated simultaneously in the same tube.Interaction between probes could be avoided easily when multiplex SNPs typing was done.3,Using AmpFl STR(?) Identifiler(?) PCR Amplification Kit and optimizing PCR conditions with 34 cycles,all STR loci could be discriminated when the starting DNA was not less than 100pg.Allele-drop occurred when the starting DNA was not more than 50pg.Using our LDR-HRCA method,final reaction products decreased as the amount of the starting genomic DNA decreased.Clear allelic discrimination was achieved as long as the homozygous DNA(AA or CC)was not less than 20 pg.10 samples of CC homozygosis(10pg of each) were detected.Among them,AC phenotype occurred in two,and no product in one.When 10 samples of AA homozygosis(10pg of each) were detected,AC phenotype occurred in one,CC phenotype in one,and no product in one.When heterozygous sample(AC) was detected,reliable allelic discrimination was achieved when the starting DNA was not less than 30 pg.10 samples of AC heterozygosis(10pg) were detected.CC phenotype occurred in one sample,AA phenotype in 2,and no product in one.No stutter was observed in all samples.Conclusions1,By treatment of genomic DNA with DNaseⅠ,it was reliable to control the degrees of DNA degradation.The method was able to be used to produce the heavily degraded DNA with a defined range of fragment lengths.2,Using our LDR-PCR method,we successfully discriminated the genotypes of each target locus from highly degraded DNA with fragment sizes less than 100 bp. Meanwhile,multiplexed allelic discrimination of all 4 SNPs was achieved in a single tube.This LDR-PCR method might be valuable for forensic DNA analysis in those cases where DNA was damaged severely.3,For DNA analysis of LCN,the specificity and sensitivity of our LDR-HRCA method were prior to enhancing STR-PCR assay,indicating that LDR-HRCA method might have potential in forensic applications.4,Based on our study,we summarized the screening standards of target SNPs loci and designing principles of LDR probes.According to these principles,the specifity and sensitivity of SNPs typing were achieved when heavily degraded DNA or trace DNA was tested.In addition,high through-put SNPs typing could be achieved with the methods based on LDR.