Estabilishing The DNA Testing Method Of Sperm Separated By Laser Capture Microdissection And Its Application In The Forensic

Objective To improve the ability of a technology that can separate the sperm from themixed cells,it′s necessary to set up a method which can effectively separate fewsperms from mixed cells,a nd can obtain a single ma le DNA genoptyping.This subjectaims to establis h a method of cell dyeing and DNA extraction which is suitable for theDNA testing of sperm which captured by laser capture microd issection;a nd use thismethod to test five individ uals′sperms DNA,to know whether there′s differencebetween these five individua ls;then to test the low copy number using this method.Inorder to make the laser caputer microd issection to pla y an important role in theforensic rape case ,this study evaluated the ability of laser capture microd issectionseparating the sperm from different rations of mixture samples which were preparedwith sperm cells and vagina l swabs under controlled conditions.Method We captured the same ma n′s sperm cells in two groups , which wererespectively stained by two dyeings(Harris'Haema toxylin and four iodine fluoresce ntred),the number of each group was 400 ,used the same extracting method and thesame conditions of PCR,each dyeing have three parallel test.By comparing the rationof loci detected,selecting a better dying which is litter impact on the test of spermDNA.We captured the same ma n′s sperm cells which stained by Harris'Haema toxylinin three groups , which were respectively extracted by three kits :DNA IQ^TM System,QIAamp○R Mini Extraction Kit and Picopure^TM DNA Extraction Kit, the number ofeach group was 400, and each kit have three parallel test.By comparing the ration ofloci detected,choosing a best kit which can effectively extract the DNA of spermDNA that separated by laser capture microd issection.We captured five differentindivid uals'sperm cells ,the number of each individ uals was 400,they were all testedby the same dyeing and the same extracting mothods established above, PCR in thesame conditions, in order to know whether there′s difference between these fiveindivid uals.Control DNA 9947A was dituled to 0.1ng/μL,0.075 ng/μL,0.05ng/μL,0.025ng/μL,0.01ng/μL,these different DNA concentrations are all less tha n 100pg,theheat cycles of 28,30,32,34 are used to PCR,select a heat cycle suitable for LCN by comparing the STR tying.Use of the above method ,and increasing the cycles to 34,thenumbers of sperm which are 30,20,10,5,4,3 were detected .Nine different rations ofmixture samples were prepared with sperm cells and vagina l swabs under controlledconditions,a nd used LCM to separate the sperms (the number all less tha n 200) fromthe mixed on the slides , extracted the sperm′s DNA and test the STR genotyping,bycalculating the ration of tested sample to improve the superiority of LCM which caneffectively separate little sperms from epithelia l cells ,and can be obtained a singlema le STR genotyping.Rusult There's clearly statistica l difference in the ratio of loci detected betweenHarris'Haema toxylin and four iodine fluorescent red.Use of Harris'Haema toxylin canobtain STR genotyping of all loci,where four iodine fluorescent red can obtain nothing.DNA IQ^TM System Kit is the best kit for extracting DNA in the three kits.Comparedthe ration of loci tested between five individ uals,a nd the statistica l results showed thatthere′s no obvious difference between this five individ uals used the mothodabove.Low copy number was tested,the sperm of 30,20,10,4 can be dectected allSTR genotying by LCM,whereas five sperms had the phenomena of drop-out,andthree sperms had the phenomena of drop-in and drop-out.LCM was used to capturedifferent numbers which all less tha n 200 from nine different ration of mixed,theration of dectected sample is 88.9﹪,and the STR genotyping are all single ma le STRgenotyping.Finally,LCM was applied to actual rape cases Successfully.Conclusion This study successfully established a method of cell dyeing and DNAextraction which is suitable for the DNA test of sperm which separated by lasercapture microd issection; evaluate the ability of laser capture microd issectionseparating the sperm from different rations of mixture samples and obtained singlema le typings ,in order to offer a basic methodology for LCM used in the forensicactual cases.