Use of bioinformatic tools for Etv6-Runx1 fusion detection associated with acute lymphocytic leukemia / Utilização de ferramentas de bioinformática para detecção da fusão Etv6-Runx1 associada à leucemia linfocítica aguda

Inaldo Antônio dos Anjos Filho, Ivson Warley Tôrres dos Anjos, Manuela Eduarda de França, Jonas José da Silva, Juliana Laguzza de Oliveira Bustos Villabón, Lidiane Gomes da Silva, Nathalia Joanne Bispo Cezar

Abstract


Genetic mutations are the main causes that predispose onco-hematological pathologies. Acute lymphoblastic leukemia (ALL) is a neoplasia with high incidence in childhood and, in these patients are found mutations involving the ETV6 and RUNX1 genes. In the present study, we evaluated in silico primers previously described in literature to delimitate the translocation ETV6-RUNX1 t (12; 21) (p13; q22). Also, the in silico primers were proposed to verify efficiency and quality in the detection of this fusion through bioinformatics tools. To detect the mutation, a primer pair was developed through bioinformatics mechanisms. Along with this, another 14 primers described in the available literature were rigorously evaluated. The National Center for Biotechnology Information (NCBI) and The European Bioinformatics Institute (EBI) databases were used to locate the target sequences and identify the genes melting points. The Primer3Plus tool was used to primers design, the Oligo analyzer software was used to evaluate the aforesaid primers using the following parameters: size 18-22 nucleotides; distribution of guanine and cytosine bases 40-60%; annealing temperature 52-60 °C; maximum of single base replicates 4 bp; Delta G value in hairpins formation above -9 kcal / mole-1 and dimers -9 kcal/mole. Finally, the gadget MEGA7 and NCBI BLAST were used to evaluate the alignment and identity analysis of the primers and possible amplicons. Based on these analyses, it was possible to observe that from the 16 primers, only 7 qualified with all recommended values within the parameters. From this total, two pairs of primers presented better results through in silico analysis, giving rise for two possible amplicons that aligned adequately and, were useful at detecting the site of gene fusion. From this study, it was possible to observe the importance of in silico analysis for researches on complex diseases. Considering that from the primers design it is possible to perform a proper PCR, these analyzes are fundamental to reduce the experiments margin of error plus, the waste of time and inputs. The selected primers in this study presented parameters that indicate good results in vitro tests and can be used to delimitate the gene fusion.

 


Keywords


primers; bioinformatics; in silico; genes; ALL; ETV6; RUNX1; gene fusion.

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DOI: https://doi.org/10.34119/bjhrv3n5-113

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