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

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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