RAPID IDENTIFICATION OF BETA-GLOBIN STRUCTURAL MUTATIONS BY SEQUENCING THE MESSENGER-RNA FROM PERIPHERAL-BLOOD RETICULOCYTES

A simple and rapid method for the molecular detection of beta-globin s tructural mutations is described using a reverse transcription-polymer ase chain reaction of reticulocyte mRNA and direct sequencing of the p roduct. The amplified segment (employing a sense primer 5'-ATTTGCTTCTG ACACAACTGT-3', located at position +1 with respect to the Cap site and an antisense primer 5'-TCCAGATGCTCAAGGCCCTTC-3', located at position +1772 with respect to the Cap site) encompasses the cDNA sequence incl uding the three globin exons. Employing this method we were able to ch aracterize two hemoglobin structural variants: Hb S (beta 6 (A3) Glu-V al: GAG-GTG) and Hb Porto Alegre (beta 9 (A6) Ser-Cys: TCT-TGT). The a pproach described in this paper should be very useful to detect hemogl obin structural variants because the RNA extraction is simple, rapid a nd does not require cesium chloride, guanidinium and proteinase K. In addition, the direct sequencing of the RT-PCR product permits the scre ening of the entire globin genes with only two reactions.