Validação de iniciadores e otimização da PCR para estudo do gene IRS1 de humanos

Abstract

Diabetes Mellitus (DM) is a group of metabolic disorders characterized by defects in insulin secretion and activity and its complications that are usually associated with dysfunction of different organs such as heart and kidneys. Among types of DM, type 2 is the most frequent in the population, it has polygenic inheritance pattern where there is also the interaction of environmental factors. One of the triggering causes of DM2 may be the disruption of insulin signaling pathways. Genetic studies in different populations show that changes in the IRS1 gene that produces the insulin receptor substrate 1 and creates molecule recognition sites for the intracellular transport of glucose may alter this signaling. PCR is a technique of molecular biology that amplifies a specific fragment of DNA and therefore enables the study of genes. Factors such as primer sequence, annealing temperature, magnesium salt concentration and template DNA interfere with the efficiency of PCR. The objective of this work was to design and validate primers, as well as to standardize amplification protocols for the study of the IRS1 gene in humans. The construct of the primers was made from a Reference Sequence for the gene under study available on the GenBank platform. Three new pairs of primers were defined, analyzed by the PrimerQuest and Oligoanalyser programs. For the validation and optimization, the analysis of the ideal conditions of the PCR was carried out where the annealing temperature (Ta), the Taq DNA polymerase enzyme, the concentration of MgSO4 and template DNA were adapted from the initial protocol. PCR products were applied on 1% agarose gel in conjunction with the molecular marker DNA Ladder 1Kb and visualized on photodocumentator. The results show that the F1/R1, F2/R2 and F3/R3 pairs of primers are suitable for the study of the IRS1 gene, as well as to establish amplification protocols for the concentration of reactants and the thermocycle for each pair of initiators. The present study will allow further work on amplification and / or sequencing of the IRS1 gene in humans, contributing to a better understanding of the genetics of DM2.