Acta bioquímica clínica latinoamericana
versão impressa ISSN 0325-2957
MARTINEZ-LOPEZ, Miriam Carolina et al. CG-rich genes amplified with the formamide, bovine seric albumin y dimethylsulfoxide mix. Acta bioquím. clín. latinoam. [online]. 2012, vol.46, n.2, pp. 285-292. ISSN 0325-2957.
Several chemical additives have been used to assure polymerization in CpG islands.The aim of the present work was to design a PCR enhancer mixture in order to amplify GC-rich genes. Fragments of IRS2 and HNF1a genes were analyzed using EMBOSS CpG Report Software. Primers were designed with the Primer3 Software and were tested with ePCR Software. Three additives were used: BSA (0.1µg/µL), DMSO (5%) and formamide (5%), in five PCR assays, two using one additive, two combining two additives and one with all additives. DNA sequences were amplified with the following enzymes: Native Taq, recombinant Taq and platinum Taq DNA polymerase. Amplicon quality was examined by sequencing. HNF1a gene was amplified without additives; however, the sequences were not amplified and showed purity problems in sequencing. The gene fragments IRS2 with CpG islands were amplified with additives DMSO, BSA and Formamida mixture, notwithstanding the enzyme used. These sequences were clean. DMSO-BSA-Formamide mixture can be a solution to obtain GC-rich DNA amplicons with such a high quality that it generates neat chromatograms during sequencing.
Palavras-chave : CpG island; Chemistry enhancer polymerase chain reaction; Insulin receptor substrate 2 gene; Hepatic nuclear factor 1a gene.