The Primary Study Of DYZ1 Array For Distinguishing Monozygotic Twin Males

Objective: Distinguishing monozygotic twin(MZT)has always been an unsolved problem in forensic case.It is not rare to see such paternity cases and criminal crime MZT involved,for example,serious jewelry theft in Berlin,German in 2009,and a 17-year-old girl being raped and murdered in UK in 2011,as well as series of 6 sexual assault cases in Marseilles of France in 2013.In these cases,the use of conventional forensic individual identification systems in these cases could not distinguish identical twins,so it is imminent to find genetic markers for effective screening of identical twins.Although several studies have manifested that DNA methylation might be an effective marker for distinguishing MZT,the instability and tissue differences of DNA methylation might limit its application in forensic cases.MPS platforms could find a few sequence differences like copy number variation(CNV),single nucleotide polymorphism(SNP)and frequent insertions and deletions polymorphism(InDel)within MZT pairs,although searching them like finding a needle in the haystack.In addition,it was reported that many SNPs and In Dels were found amongst all the three sets of MZT within DYZ1 array.DYZ1 array located in heterochromatic region of the human Y chromosome,which was identified consisting of plenty of pentameric repeat “TTCCA” and not participating in recombination,so de novo changes after birth would be recorded loyally and passed on from generation to generation.Therefore,we intended to discover whether DYZ1 array could be an effective marker to distinguish MZT individuals.In this study,we performed a subsection-amplied cloned-sequencing strategy to examine 40 sets of male MZT in the fake of attaining DYZ1 sequencing information,combined with real-time PCR(qPCR)for CNV status of DYZ1 array.Methods:1 Sample collection and DNA quantification: Our lab had built a twin sample bank in 2012 supported by Natural Science Foundation of China(NSFC).In this study,we picked out DNA samples extracted from whole blood of 41 pairs of male MZT,using Nano Q detector to test the quality and quantity of DNA.2 Detection of copy numbers: The copy numbers of DYZ1 were examined and calculated based on absolute quantification method using qPCR(GoTaq? qPCR Master Mix,Promega)on Sequence Detection System 7500 platform.The recombinant plasmids containing 3564 bp DYZ1 fragment were used as standard to build the standard curve by 10 fold dilutions of recombinant starting with 2×108 copies.Nuclease-free water and female samples were used as negative control.Each sample was repeated three times to ensure the accuracy of the results.Data were analyzed by Wilcoxon rank test using Excel and SPSS v21.0.3 Sanger sequencing of DYZ1 array: The whole 3564 bp DYZ1 fragment was amplified in four sections respectively by long Taq polymerase.The amplified products were recycled and purified to insert into a plasmid using CloneJET PCR Cloning Kit(Thermo,USA)according to instruction manual.The plasmid contained inserted DYZ1 fragment was immediately transformed into DH5α competent cells and cultured at 37℃ overnight.Then the single clone was picked up for PCR and validated by digestion of Bg1Ⅱ.Finally the positive colonies were expanded cultured in 3~5 ml LB medium,and the plasmid DNA was recovered by conventional plasmid kit.DNA of samples and standard were amplified at the same conditions,followed by purification using alcohol/EDTA,and finally loaded on sequencer with Hi-Di formamide.After quality control,the sequence was aligned with referenced sequence(X06228),and four fragments of each individual were spliced into a complete DYZ1 array by BLAST on NCBI.The sequences of identical twins are compared with each other,and the variants were marked.Results:1 CNV analysis of DYZ1 array Methods:1.1 Experimental efficacy and specificityUsing nuclease-free water and female DNA sample as negative control,the melting curve presented a single low peak at about 72℃(sample’s melting temperature was about 77℃),indicating that there was no aerosol contamination,system contamination and sequence interference of non-Y chromosomes.It was also proved that the experimental primers were specific for target DNA.In five absolute quantitative experiments,the amplification efficiency were between 95.69% ~ 100.43%,R2 were greater than 0.98,the slope was fluctuating about-3.3,and the melting curve of all samples were towering single peak.It proved that the experimental data were valid and reliable.1.2 CNV results analysis of 40 pairs of MZTThe DYZ1 copy numbers of 40 MZT pairs was detected.Except for the No.40 MZT pair,the copy numbers of DYZ1 arrays of the rest 39 MZT pairs ranged from 1141 to 9282 copies,most of them were 2000~5500 copies and the mean copy number was 5190.27 ± 162.14.Every pair of MZT showed difference of DYZ1 copy numbers,called D value,which ranged from 13 copies to 4241 copies.Among them,D values of 22 MZT pairs were larger than 1000 copies.According to Wilcoxon rank test,the D values of 19 MZ pairs(No.21~39)were statistically significant(P<0.05),accounting for 48% of total samples.For the No.40 MZT pairs,one of them showed remarkably high copy numbers of 15382.09 ± 381.04 copies that was far from the overall average,while another individual presented 5125.00 ± 818.98 copies,which was obviously lower than that of his twin brother.In summary,half of 40 detected male MZT pairs showed significant difference of CNV between each other,and another half of them had no difference.2 Sequence variants analysis of DYZ1 array2.1 Overall sequencing results40 pairs of MZT monoclonal sequencing results were obtained successfully and joined into DYZ1 array completely.The sequencing results showed that the DYZ1 length of 40 pairs of MZT was in the range of 3504 bp to 3589 bp.Comparison within each pair of MZT indicated that the matched identity was 95% ~ 99%,the number of 95%,96%,97%,98%,99% were 1,5,16,16 and 2 respectively.2.2 Four types of variants analysis in DYZ1 array of 40 MZT pairsLength difference of DYZ1 array was observed within 39 pairs of MZT except for the No.17 MZT pair,ranging from 1 bp ~ 59 bp.All of the 40 pairs of MZT showed DYZ1 sequence variants between each other.We classified the sequence variants to four types: single nucleotide polymorphism(SNP),multiple nucleotide variants(MNV),single nucleotide In Del(SNInDel),and multiple nucleotides InDel(MNInDel).Totally,the number of SNPs and MNVs were more than In Dels.For all of the examined samples,length variation totally reached to 848 bp,while sequence variation reached to 2067 bp.SNPs existed within each pair of MZT accumulated to 1882 bp,the least one being 31 bp and the most one being 67 bp.MNPs were observed in 35 pairs of MZT,most of them were two bases variants.The occurrence of SNInDel within MZT was far less than that of SNPs,totally accumulated to 187 bp in all samples.There were 21 bp and 23 bp of SNInDels in the No.27 and No.8 MZT pair respectively.The remaining pairs of MZT showed less than 10 bp of SNInDels,which was obviously less than SNPs.MNInDel mainly occurred in the form of 2 bp,3 bp,4 bp insertion and deletion,some of them expressed as more than 15 bp fragment InDel.2.3 Accuracy verification for Sanger sequencingTo validate accuracy of sequencing results,10 DNA clones were randomly selected to be sequenced repeatedly.In view of effective read range in 3730 platform(51 bp to 750 bp),two sequencing results of the same sample were identical,indicating that the accuracy of monoclonal sequencing results was 100%.Conclusions:In this study,DYZ1 arrays were sequenced and analyzed by fragment amplification,cloning and Sanger sequencing.We found that there were many sequence differences existed within all of the 40 examined pairs of MZT,including base variation and InDels.Meanwhile,20 pairs of MZT also showed CNV differences of DYZ1.In conclusion,DYZ1 array might be used as a candidate marker for discriminating male MZT and deserve further research.