| Objective:In this study,the feasibility of Chelex-100 combined adsorption column method for free edge of human fingernail extraction was verified through experiments.The effects of various factors on DNA extraction and STR genotyping of free edge of fingernails were analyzed and the optimum conditions for extracting free edge of fingernails were established.The feasibility was verified by a large number of sample experiments under the optimal extraction conditions.The content of free edge DNA of fingernails was quantitatively analyzed by analyzing the DNA concentration data of a large number of samples to determine its content.Methods:The free edge DNA of fingernails was extracted by deionized water and 10%SDS buffer cleaning combined with sterile scalpel scraping.The free edge DNA of fingernails was extracted by Chelex-100 and adsorption column.The free edge DNA of fingernails was amplified and genotyped by 5-color fluorescent Powerplex(?) 21 System PCR kit,96-well Veriti PCR amplifier and ABI 3100-Avant gene analyzer.The drying time(5mim,7min,10mim,12mim,15mim,17mim),digestion time(2h,6h,10h,14h,18h,24h),weight(3mg,10mg,14mg,15mg,16mg,17mg,18mg,19mg,20mg),thickness(0.35mm,0.40mm,0.45mm,0.50mm,0.55mm,0.60 mm),Chelex-100 concentration(1%Chelex-100,2.5%Chelex-100,5%Chelex-100,7.5%Chelex-100,10%Chelex-100)were used as the influencing factors to establish the optimum extraction conditions.Extraction and genotyping of normal samples(50 samples),fingernail polish samples(6 samples)and toenail samples(6 samples)were conducted under optimal extraction conditions.The DNA concentration of the fingernail free samples was measured,calculated and analyzed using nanodrop 2000,and the correct DNA content inside the free edge of human fingernail was obtained.We used 5%Chelex-100 to extract 50 human venous blood samples(control).The DNA genotyping of 50 fingernail free margin samples was identical with that of the control group.The GeneMapperlD v3.2 gene analysis software,EXCEL 2007 and SPSS 21.0 were used for gene analysis and statistical analysis.Results:The high quality free edge DNA of fingernails was successfully extracted by Chelex-100 combined adsorption column method.The genotyping of all loci was successfully detected under optimum DNA extraction conditions.Influencing factors of drying time,DNA atlas were complete for more than 5 minutes,no alleles were.missing,and the RFU value was positively correlated with the drying time.When the drying time exceeded 15 minutes,there were more miscellaneous peaks in the TH01,vWA,D8S1179,and D12S391 loci.Therefore,the best drying time is 10 minutes.Among the influencing factors of digestion time,the 2,1,and 1 alleles were deleted at 2h,6h,and 10h,respectively.At 2h,there was also an unbalanced peak at the Amelogeninlocus.At 14h,the locus Penta E and Penta D were not equilibrium peak appeared.At 18 hours and 24 hours,there were no alleles loss found and allelic peaks were well-balanced.24h locus gene had the highest peak RFU value,24h was the best digestion time.Among the weight influencing factors,the weight was 3mg,10mg were missing 17 and 12 alleles.The gene map above 14mg was intact,the peak type was stable.Among the thickness influencing factors,there were 5 and 1 allele deletions in the 0.35mm and 0.40mm thick fingernail samples respectively,and a complete pattern began to appear at 0.45mm.Amonng the factors affecting the concentration of Chelex-100,22 and 16 alleles were deleted in the DNA-STR typing map at the concentration of 1%and 2.5%,respectively.The concentration of Chelex-100 was above 5%,the DNA-STR genograph was complete and no allele.The DNA concentration extracted by 7.5%Chelex-100 was the highest,and the STR typing pattern was almost the same as that of 10%Chelex-100.Therefore,when the concentration of Chelex-100 was 7.5%-10%,the best one was obtained.50 samples of nail free margin samples,50 blood samples(control),6 samples of fingernail polish and 6 toenail samples were successfully extracted from DNA and STR genotyping was performed under the optimal conditions.The amount of DNA in free margin of fingernail is about28.15 ±5.81ng/mg.Conclusion:Chelex-100 combined adsorption column method is feasible and suitable for DNA extraction from fingernail free edge.Through this study,the final optimal conditions for DNA extraction are determined as follows:the weight is more than 14 mg,the thickness is more than 0.45mm,the digestion time is 24 hours,the temperature is 100? and the concentration of Chelex-100 is 7.5%-10%.The results of this study provide valuable experimental basis for forensic identification of free edge DNA of fingernails. |
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