Forensic Assessment Of STR Typing And MtDNA Sequencing In Carious Lesion

Background:Human teeth are biological structures that resist extreme conditions thus a useful source of DNA for human forensic identification.However,previous forensic identification depended mainly on the pulp and other hard tissues of intact teeth.In some situations,only carious teeth are available for forensic analysis.The caries are known to destroy enamel and dentin and also the pulp may also undergo necrosis due to caries.As the pulp tissues are major source of DNA in forensic evaluation,implications of caries needs to be evaluated.Objective : The aim of this study was to establish a DNA extraction technique from human teeth,which is valid for carious teeth,cost-effective,time-saving,and conserves the tooth integrity and to validate STR profiling from carious teeth.Methods:120 carious human teeth were collected during 2016-2018 the First Affiliated Hospital of China Medical University(Shenyang,China).Each patient signed a consent that was approved by the ethical review committee of China Medical University,Shenyang,Liaoning Province,P.R.China.Carious teeth were extracted under strict sterilized conditions by professional dental surgeons and stored into sterilized gauze until further process.The inclusion criteria were the presence of carious pathology diagnosed either radiographically or visually,whereas exclusion criteria were root canal treated teeth,internal root resorption,periapical pathology,and severe open root apex.The teeth were decontaminated by gentle bleaching and complete soft tissue curettage and then preserved at-20? until being used.Collection of carious lesion samples was performed by a skillful dentist,as per International caries detection and assessment system(ICDAS)classification,radiographic images and whether soft or hard carious lesion was present.The superficial debris and plaque were removed by reduced transport fluid(RTF;Syed & Loesche,1972)two times.We compared two DNA extraction procedures on carious teeth listed below:Operative technique:(40 molars,11 premolars,and 9 incisors).We used sterile diamond bur cut around hard carious lesion and hand excavated soft carious dentine with sterile spoon excavators of different sizes.The collected hard and soft dentine caries fragments were preserved in micro-centrifuge tubes and analyzed separately.Crushing was needed for a few samples where hard carious enamel was collected.Cervical cut:(45 molars,7 premolars,8 incisors)A cut at the neck level of the carious tooth using tungsten carbide surgical bur facilitates direct access to the pulp.Pulpal tissue samples were collected using endodontic K-files of different sizes from the pulp chamber,root canals,and the dental apex of the root(Fig.3).In both groups,6-20 gm of samples was collected in microcentrifuge tubes.PCR coamplification of one sex locus and 19 autosomal STRs,including 13 combined DNA index system(CODIS)STRs and 6 other loci were performed in a fluorescence-based multiplex reaction using the Goldeneye 20 A systems.Analysis of results was obtained in4 different possibilities: 1)Full profile(where all 19 STR loci markers and amelogenin gene was present),2)Partial profile(more than 10 STR loci could be identified and amelogenin gene),3)Low profile(less than 10 STR loci could be identified and amelogenin gene),4)No profile(no STR loci could be identified,whether amelogenin gene could be identified or not).Random 12 samples from the low and partial profile STR results undergone mitochondrial DNA sequencing in both hypervariable regions 1and 2,both in forward and reverse primer.Results:All participants included in the current study were from the same geographical area and the same dentist performed dental extractions.Total 37 females and 23 males participated in the operative cut technique group,whereas 42 females and 18 males participated in the cervical cut technique group.The age range was of participants was15-68 in the operative cut group,whereas the age range was 17-64 in the cervical cut group.The operative technique showed a conservative approach to the sampling of carious tissues and allowed safe access to collect carious tissues,whereas cervical cut technique permitted access to the root canals and complete sampling of pulp tissues.The process of DNA extraction was successfulin both groups,purity(A260/280)values were symmetric,yet nucleic acid concentrations showed wide variations.Of the 120 samples,118(98%)samples obtained genetic profiles that could be used in forensic identification.Twelve random samples,where 3 low profile and 3 partial profile STR results from operative cut technique and cervical cut technique respectively and with various nucleic acid concentration have been tested for mitochondrial DNA sequencing in both HV1 and HV2 and showed good sequencing results similar to the reference DNA sample.Conclusions:1.Operative technique by drilling holes on the defected surface of carious human teeth and gentle hand excavation of carious tissues would be used in recovery of DNA from carious teeth.2.Operative technique is efficient,preservative,time saving and cost effective method.3.Soft and hard carious material could be used for forensic identification purposes.