HOMOGENEITY OF THE Hb LEPORE GENE IN FR YUGOSLAVIA
Urosevic J1, Djureinovic T1, Poznanic J1, Cvorkov-Drazic M2, Bunjevacki G2, Janic D3, Krivokapic-Dokmanovic L3, Popovic Z1, Pavlovic S1
*Corresponding Author: Dr. Sonja Pavlovic, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, 11000 Belgrade, FR Yugoslavia; Tel: +381 11 3976 445; Fax: +381 11 3975 808; E-mail: sonya@sezampro.yu
page: 29

MATERIAL AND METHODS

Eighteen Hb Lepore carriers from six unrelated families were studied. Hematological parameters were obtained using an automated cell counter. Hb F was quantitated by standard methods [11,12] and Hb A2 was estimated by elution from celogel electrophoretic strips. The abnormal Hb was detected by electrophoresis on cellulose acetate in Tris-borate-EDTA (pH 8.4) [13]. Genomic DNA was extracted from peripheral blood collected with sodium-citrate as an anticoagulant [14].

DNA samples were used as templates in polymerase chain reaction (PCR). For each sample, a single PCR was performed using primers A, B and C. A 356 bp fragment was amplified by using primers A (5-AATCATTCGTC TGTTTCCCA-3) and B (5-GTTTTCCTACCCTCAGA TT-3). With these two primers only fusion db genes can be detected. A 751 bp fragment was amplified by using primers A and C (5-CCAATCTACTCCCAG GAGCA-3). This fragment can be obtained only in the presence of a normal b-globin gene.

Finally, the sequence of a 356 bp fragment was determined by the dideoxy chain termination method using fluorescently labeled dideoxy nts (Pharmacia Biotech, Uppsala, Sweden) on an automated DNA sequencer (ALFexpress DNA Sequencer; Pharmacia Biotech), using primers A (as forward) and B (as reverse).




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