Balkan Journal of Medical Genetics

COMPLEMENT FACTOR H Y403H POLYMORPHISM IN THE TURKISH POPULATION
Yunus Arikan Y, Türker Bilgen T, Ibrahim Keser I,
*Corresponding Author: Ibrahim Keser, Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University, TR-07059, Antalya, Turkey; Tel.:+90-242-249-6973; Fax:+90- 242-227-4495; E-mail: keser@akdeniz.edu.tr
page: 41

MATERIALS AND METHODS

We examined 54 females and 46 males (aged 17 to51 years) from the university staff, who declared that they had no health problems. They were all from Antalya Province in Turkey and all gave their informed consent. Genomic DNA was isolated from whole peripheral blood samples by the classic saltingout procedure [19]. The region which includes the c.1277 T>C variant in the CFH gene was amplified using polymerase chain reaction (PCR) by forward primer 5’-CTT TGT TAG TAA CTT TAG TTC GTC TTC AG-3’ and reverse primer 5’-CAA GGT GAC ATA ACA TTT TGC C-3’. Approximately 100 ng of genomic DNA, measured by NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA), was used in 25 μL reaction volumes. The PCR mixture contained: 1.25 U Taq polymerase (GoTaq; Promega, Madison, WI, USA), 10X buffer (pH 8.8 with ammonium sulfate; Fermentas, Vilnius, Lithuania), 1.5 mM MgCl2 (Fermentas), 5 pmol of each primer and 200 mM of each of the four dNTPs. The PCR program consisted of 40 cycles of initial denaturation at 93°C for 150 seconds, 36 cycles of 30 seconds at 93°C, 45 seconds at 58°C, 60 seconds at 72°C, and final extension at 72°C for 5 min. The 443 bp PCR products were electrophoresed on 2.5% agarose gel containing ethidium bromide, and then visualized under UV light using imaging system (SynGene InGenius, Cambridge, Cambridgeshire, UK). The PCR products were purified by PureLink™ PCR Purification Kit (Invitrogene, Carlsbad, CA, USA). Sequencing was done using the BigDye Terminator v3.1 Cycle Sequencing kit and an ABI PRISM™ 3130 × l Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The allele and genotype frequencies were calculated using the allele counting method.

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