Download 6.1. need for the study - Rajiv Gandhi University of Health Sciences

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
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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”
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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.
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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:
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
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.
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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
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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
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
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
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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
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