文献_Detection of unknown deletions in β-globin gene cluster using relative quantitative PCR methods.pdfVIP

European Journal of Haematology 83 (261–269) ORIGINAL ARTICLE Detection of unknown deletions in b-globin gene cluster using relative quantitative PCR methods 1,2,3 1 1 1 1 Sadegh Babashah , Somayeh Jamali , Reza Mahdian , Mina Hayat Nosaeid , Morteza Karimipoor , Raheleh Alimohammadi2,3 2 1 2 , Marzieh Raeisi , Fereshteh Maryami , Mahboubeh Masoudifar , Sirous Zeinali1,2 1Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; 2 Kawsar Human Genetics Research Center, Kawsar Genomics Center, Tehran, Iran; 3 Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran Abstract b-Thalassemia is mainly caused by mutations involving single base substitution and small deletions. How- ever, a considerable number of carriers are suspected to have large deletions in b-globin gene cluster. Common strategy for identifying deletions with definite breakpoints is based on Gap PCR. There are, how- ever, some cases with indefinite breakpoints which usually cannot be detected by this method. We devel- oped and optimized a quantitative real-time PCR assay for copy number analysis of b-globin gene cluster. The copy number of target fragments (i.e. b, d or Gc-globin genes) was determined using comparative threshold cycle method. In addition, gene dosage was analyzed using multiplex ligation-dependent probe amplification (MLPA) method in all suspected carriers. Using these relative quantitative assays, normal or carrier statuses of all 26 unknown sam