Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Marmara Pharmaceutical Journal 1990 , Vol 6 , Issue 1
Investigations of some point mutations in beta thalassemia patients by gene amplification and synthetic oligonucleotide probe hybridization methods
Oya Uyguner,Turay Yardımcı,Engin Bermek,Beyazıt Çırakoğlu,Muzaffer Aksoy
In this study, the DNAs of one healthy individual and nine patients with anemia or beta thalassemia were analized to detect some of the point mutations which may cause beta thalassemia.

The DNA was isolated from approximately 5 ml of blood and 0.5 mg of that DNA was amplified in a reaction mixture containing primers bordering the related area in the β-globulin gene, deoxyribunecleotide (dNTPs), and taq polymerase enzyme. The amplification was done by 25 subsequent cycles consisting of denaturation at 95ºC for one minute, primer hybridization at 55ºC for one minute, and primer extention at 72ºC for three minutes. Amplified DNA samples were transferred onto nylon membrane at equal amounts after the concentrations were adjusted by agarose gel electrophoresis, and hybridized with four different 19 nucleotide (nt) long synthetic oligonucleotide probes containing point mutations, which were labelled with 32PdATP at 5’ ends. It was found that the three patients had GA at IVS-1 nt 1, two patients had TC at IVS-1 nt 6, and one patient had CA point mutation at exon 2 codon 39. Since each of these six samples were hybridized both with the normal and mutant probes, they were identified to be heterozygote for these mutations. None of the samples showed GA mutation at IVS-1 nt 110. Normal (healthy) sample hybridized with only normal probes and did not hybridize with any of the mutant probes. Keywords :

Marmara University