FREQUENCY OF THE CFTR 2694T/G POLYMORPHISM AND ITS ASSOCIATION WITH CFTR-RELATED MONOSYMPTOMATIC DISORDERS
Nikolic A, Divac A, Kusic J, Savic A
*Corresponding Author: Dr. Aleksandra Nikolic, Laboratory for Molecular Biology, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P. O. Box 446, 11001 Belgrade, Yugoslavia; Tel: +381-11-397-6658; Fax: +381-11-397-5808; E-mail: qwert@eunet.yu
page: 43

MATERIAL AND METHODS

DNA was isolated from peripheral leukocytes using standard methods [4]. Polymerase chain reaction (PCR) amplification of exon 14a, using primers already described [5], was carried out in a 25 mL volume containing 1x Taq buffer (50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100; Promega, Madison, WI, USA), 2.5 mM MgCl2, 0.2 mM dNTPs, 5 pmol of each primer, 1 U of Taq polymerase (Promega) and 100-200 ng of DNA. PCR conditions were as follows: initial denaturation at 94C for 5 minutes, 40 cycles of 1 minute denaturation at 94C, 1 minute annealing at 55C, 2 minute extension at 72C, with final extension of 10 minutes at 72C.


The PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) analysis in 6.5% polyacrylamide gel containing linearly increasing denaturing gradient formed by urea (0.7-4.2 M) and formamide (4‑24%). Electrophoresis was followed by silver staining of the gel [6].




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