The mitochondrial DNA (mtDNA), the second genetic system of the eukaryotic cell, plays an important role in the cellular physiology and is frequently involved in the development of pathologic processes. Its small size (16.569 bp) makes the mitochondrial genome susceptible of being completely analyzed in order to identify sequence variations that result in functional anomalies. However, there were no strategies to sequence completely the mtDNA at low cost. A side project of Dr Esther Gallardo Perez during her studies on mitochondrial genetics in our laboratory was to develop a simple, fast and affordable protocol to completely sequence and annotate the mtDNA on a routine basis. Nowdays, Secugen SL is commercializing this procedure to diagnose mitochondrial diseases.