Estandarización de la reacción en cadena de la polimerasa (PCR) específica para la amplificación del fragmento rs35532010 presente en el gen de la beta globina

Abstract

Thalassemias are hereditary diseases resulting from genetic defects that cause an alteration in the synthesis of globin chains. The β-thalassemia encompasses a group of mutations that cause defects in the β-globin chains. The 27/28 +C mutation, is characterized by the insertion

of a cytosine nucleotide that causes a change in the reading frame of the gene resulting in β- thalassemia. Although few cases have been reported in countries where the prevalence of β- thalassemia is very high, information on this mutation in Latin America is scarce. However,

unpublished data have identified this mutation as prevalent in the Ecuadorian population. The main objective of this titration work was to standardize a PCR protocol for the amplification of the rs35532010 polymorphism of the hemoglobin beta subunit (HBB) gene. Commercial DNA samples and specific primers were used to amplify the fragment of the gene where this mutation is located. Three different protocols were performed for standardization, being the first assay the one that allowed establishing the ideal conditions of 1μl of DNA diluted at 80ng/μl, at an alignment temperature of 64oC, for fragment identification by electrophoresis in 2% agarose gel. However, the use of polyacrylamide gel, of higher resolution and wide separation range, is recommended to improve the visualization of the bands. Standardization of this protocol will be of great utility for subsequent sequencing to determine allelic zygosity status in patients with β-thalassemia.