Faculty of Biotechnology and Food Sciences in Nitra Journal of Microbiology, Biotechnology and Food Sciences 1338-5178 no. 5 vol. 6 2017-04-03 2017-04-03 REVISITING MOLECULAR CLONING TO SOLVE GENOME SEQUENCING PROJECT CONFLICTS 1157 1160 EN Hugo A. Barrera-Saldaña Hugo A. Barrera-Saldaña Aarón Daniel Ramírez-Sánchez Aarón Daniel Ramírez-Sánchez Tiffany Editth Palacios-Tovar Tiffany Editth Palacios-Tovar Dionicio Aguirre-Treviño Dionicio Aguirre-Treviño Saúl Felipe Karr-de-León Saúl Felipe Karr-de-León In our laboratory, DNA sequencing by Sanger method is used as the “gold standard” for clinical diagnostics, microbe identification (bacteria and yeast, mainly) and genome characterization. In this research, we used it to characterize a conflicting locus in a Saccharomyces cerevisiae sequencing project. When sequenced, the resulting electropherogram of the analyzed locus showed a pattern indicating either sample contamination or allele variation. Molecular cloning was chosen as the most straight-forward strategy to solve the dilemma. The initial characterization of recombinant plasmids by restriction enzyme digestion confirmed the presence of two genomic sequences. Their Sanger sequencing revealed two alleles distinguishable by a total of 29 nucleotide differences (25 of which were SNPs). NCBI BLAST revealed that the conflicting locus covered an intergenic region and a coding sequence for a putative permease protein. The present study shows the utility of the classical molecular cloning technique to solve problems of modern genome projects.
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