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We determined the spectrum of beta-thalassemia (thal) mutations in 118 affected unrelated patients with different forms of beta-thal. Using a combination of reverse dot-blot analysis, denaturing gradient gel electrophoresis (DGGE), polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) and direct nucleotide sequencing, we identified the largest spectrum of beta-thal mutations so far reported in Tunisia, and to the best of our knowledge, within the Mediterranean Basin. A total of 18 distinct alleles were detected at different frequencies, with two alleles [codon 39 (C-->T) and IVS-I-110 (G-->A)] predominating all others. Seven other alleles [frameshift at codon (FSC) 6 (-A), FSC 8 (-AA), codon 30 (G-->C), IVS-I-1 (G-->A), IVS-I-2 (T-->G), IVS-I-6 (T-->C), FSC 44 (-C)] were rare, and nine alleles [-29 (A-->G), IVS-I-2 (T-->C), IVS-I-5 (G-->C), IVS-I-5 (G-->T), IVS-I-116 (T-->G), codon 37 (G-->A), IVS-II-1 (G-->A), IVS-II-745 (G-->C) and IVS-II-849 (A-->C)], albeit described elsewhere, are reported here in Tunisia for the first time. The codon 39 and IVS-I-110 mutations were the two predominant alleles occurring at frequencies of 43.8% and 10.8%, respectively. They are presumably the earliest mutations introduced into this country. The codon 39 allele could have been introduced in Tunisia during the Roman occupation. Similarly, the IVS-I-110 mutation might have been introduced by the Turkish and Phoenician influence. Both gene flow and private mutations may account for the diversity of alleles observed in Tunisia. These data provide the background for implementing prevention programs based on genetic counseling and prenatal diagnosis.

Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5' end of one of the primers, whereas the other primer starts with the nucleotide at position (-1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat alpha integrin CD11b A-domain and the human CD8beta cloned in pPICZalphaB, pGEX-2T, and CDM8 expression vectors, respectively.