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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA. ANNEXURE – II PROFORMA FOR THE REGISTRATION OF SUBJECT FOR DISSERTATION. BY DR. PRIYANJALI DUTTA 1ST YEAR MDS. DEPARTMENT OF ORAL & MAXILLOFACIAL PATHOLOGY 2010 1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. NAME OF THE CANDIDATE AND ADDRESS: DR. PRIYANJALI DUTTA POST GRADUATE STUDENT IN DEPARTMENT OF ORAL & MAXILLOFACIAL PATHOLOGY, KRISHNADEVARAYA COLLEGE OF DENTAL SCIENCES & HOSPITAL, KRISHNADEVARAYA NAGAR, HUNASAMARANAHALLI VIA, YELAHANKA, BANGALORE 562157. KRISHNADEVARAYA COLLEGE OF DENTAL SCIENCES & HOSPITAL. 2. NAME OF THE INSTITUTION: 3. COURSE OF THE STUDY AND MASTER OF DENTAL SURGERY ORAL & MAXILLOFACIAL SUBJECT: PATHOLOGY 4. DATE OF ADMISSION TO COURSE 5. TITLE OF THE TOPIC 03-05-10 “RAPID AND SIMPLE SEX DETERMINATION FROM DENTAL PULP TISSUE BY CONVENTIONAL POLYMERASE CHAIN REACTION TECHNIQUE” 2 6. BRIEF RESUME OF INTENDED WORK 6.1. NEED FOR THE STUDY: In the event of any mass fatality incident, despite the cause , disaster victim identification must be undertaken. DNA plays a vital role in disaster victim identification. [1] Human identification is one of the major fields of study and research in forensic science because it deals with the human body and aims at establishing human identity. [2] Teeth have a rich supply of genetic information. [5]They are the prime choice for extracting DNA for identification of individuals in mass disasters as teeth are the one of the most resistant structures in body to desiccation and thus are a better source of DNA. [4] Determining the sex of a given DNA sample from either dental pulp or dentin of tooth can also provide criminal investigators with useful intelligence and can aid the identification of missing persons and disaster victims [3] PCR based systems that amplify regions of amelogenin gene have become the method of choice for sex determination of biological samples. [3] The purpose of the study is to determine the gender of the person using dental pulp subjected to different environmental insults as a source of DNA using conventional PCR technique , as a substantial amount of DNA can be obtained from this tissue. 3 6.2. REVIEW OF LITERATURE:Dental pulp is a good source of DNA in the tooth. [5] From the DNA that is extracted from the dental pulp sample, amelogenin gene locus is used for sex determination by amplifying a segment of X-Y homologous gene locus through PCR based analysis. There are two amelogenin genes, one on X- chromosome at distal short arm in the region p22.1-p22.3 and other on Y- chromosome near centromere. [6] PCR amplification of amelogenin gene acts as a reliable and rapid means of determination of sex chromosomes. [8]PCR involves three basic steps which when repeated time and again results in multiple copies of a specific target sequence. The steps are Denaturation, annealing, extension of annealed fragments. [9] The environmental influence on the concentration, integrity and recovery of DNA extracted from dental pulps has been previously measured by Schwartz et al. with varied pH and temperature for different time periods (1- 6 months). It was determined that the environmental conditions examined did not affect the ability to obtain high-molecular-weight human DNA from dental pulp. [10] 6.3. OBJECTIVES OF THE STUDY: 7. To determine the gender of the person from whole dental pulp tissue obtained from the tooth samples which have been subjected to various environmental insults, by amplifying the DNA sequences based on amelogenin gene locus using conventional PCR technique. MATERIAL & METHODS: 7.1. SOURCE OF SAMPLE: 4 Dental pulp samples are obtained from 50 tooth samples Source:o Ten freshly extracted teeth (Five – normal and five carious teeth) o Five tooth samples kept in salt water for 1 month o Five tooth samples kept in salt water for 3months o Five tooth samples kept at normal room temperature for desiccation for 1month o Five tooth samples kept at normal room temperature for desiccation for 3 months o Ten tooth samples incinerated by wrapping in cotton with alcohol and burned o Five tooth samples stored approximately 10 cm deep in garden soil for 1 month [11] o Five teeth samples stored approximately 10 cm deep in garden soil for 3 months The tooth samples after being subjected to various environmental insults are washed in 5.2 % sodium hypochlorite solution. Then the teeth are cleaned and washed again with sterile distilled water. 7.2 METHODOLOGY: Technique of deriving pulp tissue:- Conventional endodontic access: Access opening is done with the help of an aerotor handpiece and pulp tissue is retrieved. The pulp tissue obtained from one of the above methods is then put into a DNA extraction buffer(Sodium dodecyl sulphate or sodium lauryl sulphate (SDS).The DNA from the above solution was extracted using phenol-chloroform method. The acquired extract was concentrated by centrifugation and then incubated at 62οC and the supernatant is taken in a 2 microlitre pipette. 5 METHODOLOGY FOR AMPLIFICATION OF TEMPLATE DNA SEQUENCE:o PRIMER DESIGN SPECIFIC FOR THE TARGET SEQUENCE: A set of four specially designed primers namely FIP, BIP, B3 and F3 prepared on a database from gene bank to detect X and Y alleles of amelogenin locus is used. X specific and Y specific primers were stored in freezer temperature (-20οC) separately to conduct the reaction. o CONVENTIONAL PCR TECHNIQUE OF AMPLIFICATION: This method employs a Taq DNA polymerase and a set of four specially designed primers that recognize distinct sequences on target DNA. MATERIALS:-The amplification reaction products which are put in 0.2ml flat capped PCR tube consists of 1.25 U Taq polymerase, 5μl 10X buffer, 2mM MgCl2, 10mg/ml BSA, 250μM dNTPs, 200nM of each primer, 1-3 μl of the template DNA which was taken from the supernatant from the 2 microlitre pipette Distilled Water to a total volume of 50μl. ARMEMTARIUM:1. Micro-pipettes a. 200-1000ul- 1No. b. 20-200ul- 1No. c. 2-20ul- 1No. d. 0.2-2ul- 1No. 2. Micro- tips a. 200-1000ul- 2 boxes b. 20-200ul- 5 boxes 6 c. 0.2-2ul- 5 boxes d. Tip boxes ( for all volumes mentioned above) 3. Microfuge tubes a. 1.5ml - 1box b. 2ml- 1box c. 0.5ml- 1 box 4. PCR tubes a. 0.2ml flat capped- 100No.s- 1pack 5. Micropestle (needed only if have to homogenize the tissue sample in the microfuge tube) 6. 00C and 200C coolers- 1No. each (for storage of the PCR reagents while preparing for the reaction) 7. Ice float- 1No. (for keeping the PCR tubes containing samples during preparation for the reaction) PROCEDURE:The PCR reaction is as follows: Initial denaturation- 950C for 5 min Followed by 35 cycles of Denaturation- 950C for 30 sec Annealing- 550C for 30 sec Extension- 720C for 30 sec Followed by a final extension- 720C for 30 min The above PCR product was then finally visualised on a 4% metaphor gel containing ethidium bromide or 1.5% agarose gel. Inclusion criteria Tooth samples of known age (18-60 years) and gender Normal tooth samples with vital pulp which are desiccated at room temperature. Carious tooth samples with regressive alterations 7 Permanent tooth samples Exclusion criteria – Tooth samples whose gender is not known. Perforated tooth samples Grossly destructed tooth samples Completely calcified tooth samples Deciduous tooth samples EXPECTED RESULTS:- The amplification of a single pair 106 base pair segment of amelogenin gene corresponding to X- chromosome indicates the female origin of those samples. The double PCR bands on metaphor gel ( 106 base pair and 112 base pair) corresponding to X and Y chromosomes indicate male origin of the tooth samples.[12] By using this method results can be obtained in around 3-4 hrs which is lesser than conventional technique. STATISTICAL ANALYSIS:- Statistical analysis will be performed using Statistical package for social scientists (SPSS). 7.3. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR OTHER ANIMALS? NO 7.4. HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3? YES A COPY OF THE SAME HAS BEEN ENCLOSED 8 LIST OF REFERNCES:1) Eleanor A.M. Graham ; Disaster victim identification; Journal of forensic science , medicine and pathology Vol. 2, no.3, 203-207 2) Ricardo Henrique Alves da SILVA; Use of DNA technology in forensic dentistry ; Journal of Applied Oral Sci. 2007;15(3):156-161 3) Eleanor A.M. Graham; Sex determination; Journal of forensic science, medicine and pathology Vol.2, No. 4, 283-286 4) Gaytmenn R.; Quantification of forensic DNA from various regions of human teeth ; Journal Forensic Sci. 2003 May;48(3);622-625 5) I. Lijnen ; DNA research in forensic dentistry; Journal of Methods Find Exp. Clin. Pharamacol 2001, 23(9),235-242 6) Satoshi Sasaki ;The amelogenin gene; Int. Journal Dev. Biol.39, 127-133 7) AN113: Gender determination by amelogenin PCR and product analysis by DNA chromatography 8) Indian Journal of Medical Research 1998 9) PCR 2nd edition by Michael Mc Pherson and Simon Moller 10)Gajendra veerarahgavan ; Determination of sex from tooth pulp tissue ; Libyan Journal of Medicine, Vol 5 (2010) incl Supplements 11) A. Corte Real ; The DNA extraction from pulp-dentin complex of and without carious ; International congress series 1288 (2006) 710-712 both with 12) Kovatsi L ; DNA repair enables sex identification in genetic material from human teeth; Hippokratia 2009,13,3 ;165-168 9