INVESTIGATION OF THE N-TERMINAL CODING REGION
OF MUC7 ALTERATIONS IN DENTISTRY STUDENTS
WITH AND WITHOUT CARIES Koç Öztürk L, Yarat A, Akyuz S, Furuncuoglu H, Ulucan K, *Corresponding Author: Dr. Korkut Ulucan, Department of Molecular Biology and Genetics, Faculty of
Engineering and Natural Sciences, Üsküdar University, Haluk Turksoy Sok. No. 14, Üsküdar, İstanbul 34662,
Turkey. Tel: +90-216-400-2222-2409. Fax: +90-216-400-2222; Mobile: +90-532-692-1922.
E-Mail: korkutulucan@hotmail.com page: 71
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MATERIALS AND METHODS
Study Groups. The study included 24 dental students
with caries aged between 18 to 23 years (mean
age 20.2 ± 1.2) and 20 dental students without caries
aged between 19 to 26 years (mean age 21.95 ± 2.2).
Forty-four healthy dentistry students were enrolled in
this study, 24 of them (aged between 18 to23 years,
mean age 20.2 ± 1.2) were classified to have carries
[decayed, missing, filled-teeth (DMFT)=5.6] according
to the World Health Organization (WHO) criteria,
and 20 of them (aged between 19 to 26, mean age
21.95 ± 2.2) were caries- free (DMFT=0). Inclusion
criteria for subjects with caries and without caries
were as follows; no oral complains, good oral hygiene,
absence of smoking and drinking habits, no systemic
diseases, no drug abuse. Exclusion criteria was unwillingness
to participate in the study. The subjects signed
an informed consent form to participate in this study.
Clinical Examination. One experienced dentist
examined all subjects for their adherence to inclusion
criteria. The WHO criteria were used for DMFT [6].
The oral hygiene and gingival status were assessed
using the simplified oral hygiene index (OHI-S) and
gingival index (GI) [7]. A structured questionnaire
was used to collect data on oral hygiene habits (frequency
of tooth brushing, use of dental floss), professional
counseling on oral health and hygiene, and the
presence of gingival bleeding. All subjects had good
oral health and had regular dental care.
Collection of Saliva. Fasting unstimulated
whole saliva samples were collected at the same time
of day (between 08:30 and 11:00 am) for a 1-week
period by the same researcher in all cases. Before
saliva collection, the mouth was rinsed with distilled
water. Subsequently, saliva was allowed to accumulate
on the floor of the mouth and the subjects were
instructed to spit into the test tubes. During the saliva
collection, the participants stayed in a relaxed
position with their heads bent forward. Each saliva
collection period was 10 minutes long. Immediately
after collection, saliva volume was measured and
then salivary flow rate calculated as mL/min. Saliva
samples were stored at –20 °C until used.
Salivary Analysis. The temperature of the saliva
samples was raised from –24 °C to 4 °C and kept for
an hour. After thawing, they were centrifuged and
supernatants were used for salivary analysis. Total
protein level was determined by the method of Lowry
et al. [8]. Bovine serum albumin (BSA) was used as a
standard, and absorbance evaluated at 500 nm. Total
protein level was expressed as mg%. Salivary pH was
directly measured with pH paper (pH indikatörpapier,
Merck Neutrolit-5.5-9.0; Merck, Darmstadt, Germany).
Salivary buffer capacity (SBC) was measured
by Ericsson’s method, modified by Menteş et al. [9].
Two hundred μL of saliva was mixed with 600 μL HCl
(0.0033 M). After 10 min., the pH of the mixture was
immediately measured with pH paper (pH indikatörpapier,
Merck Neutrolit-5.5-9.0; Merck). Based on
the color change of the indicator paper strip, the pH
was assessed in comparison with a color chart. The
corresponding value is taken as the pH of the mixture.
DNA Genotyping. The Marmara University
Ethics Committee approved the study protocol, and
written informed consent was obtained from all participants.
DNA was extracted from peripheral blood
cells by using High Pure polymerase chain reaction
(PCR) Template Preparation Kit (Roche Diagnostics
Deutschland GmbH, Mannheim, Germany) according
to the manufacturer’s instructions. Isolated
DNA samples were kept at –20 °C if not studied on
the same day. The MUC7 N-terminal coding region
was amplified using specific primers: 5’-GAA GGT
CGA GAA AGG GAT CAT-3’ and 5’-GTC TTG TGG
AGC TGG GGA AT-3’. Polymerase chain reaction
was performed in a final volume of 50 μL containing
2-10 ng DNA, 200 mM of each deoxynucleotide
triphosphate (dNTP), 1.5 mM MgCl2, 10 mM Tris/
HCl, pH 8.3, 50 mM KCl, 10 pmole of each primer,
and 0.1 U Taq polymerase. Thermocycle settings
consisted of a denaturation step at 94 °C for 3 min.,
followed by 35 cycles at 94 °C for 60 seconds, 62 °C
for 60 seconds, and 72 °C for 60 seconds and a final
extension at 72 °C for 5 min. The 199 bp amplicons
were sequenced with ABI PRISM™ 3100 Genetic
Analyzer using DYNemic ET Terminator Cycle Sequencing
Kit (GE Healthcare Life Sciences, Little
Chalfont, Buck-inghamshire, UK).
Statistical Analysis. The significance in the statistical
analyses between groups was assessed using
the χ2 test [Statistical Package for the Social Sciences
(SPSS) 10.0 for Windows; SPSS Inc., Chicago, IL,
USA]. Relationships yielding p values less than 0.05
were considered to be significant.
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