Ashael Alfredo Pérez-Muñoz, María de Lourdes Muñoz*, Normand García-Hernández* and Heriberto Santander-Lucio Pages 151 - 164 ( 14 )
Mitochondrial DNA (mtDNA) methylation has the potential to be used as a biomarker of human development or disease. However, mtDNA methylation procedures are costly and time-consuming. Therefore, we developed a new approach based on an RT-PCR assay for the base site identification of methylated cytosine in the control region of mtDNA through a simple, fast, specific, and low-cost strategy. Total DNA was purified, and methylation was determined by RT-PCR bisulfite sequencing. This procedure included the DNA purification, bisulfite treatment and RT-PCR amplification of the control region divided into three subregions with specific primers. Sequences obtained with and without the bisulfite treatment were compared to identify the methylated cytosine dinucleotides. Furthermore, the efficiency of C to U conversion of cytosines was assessed by including a negative control. Interestingly, mtDNA methylation was observed mainly within non-Cphosphate- G (non-CpG) dinucleotides and mostly in the regions containing regulatory elements, such as OH or CSBI, CSBII, and CSBIII. This new approach will promote the generation of new information regarding mtDNA methylation patterns in samples from patients with different pathologies or that are exposed to a toxic environment in diverse human populations.
Mitochondrial epigenetics, bisulfite sequencing, methylation, mitochondrial DNA, D-loop, RT-PCR.
Department of Genetics and Molecular Biology, Research and Advanced Studies Center of National Polytechnic Institute (CINVESTAV of IPN), Mexico City, Department of Genetics and Molecular Biology, Research and Advanced Studies Center of National Polytechnic Institute (CINVESTAV of IPN), Mexico City, Unidad de Investigacion Medica en Genetica Humana, Unidad Medica de Alta Especialidad Hospital de Pediatria “Dr. Silvestre Frenk Freund”, Centro Medico Nacional “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Department of Genetics and Molecular Biology, Research and Advanced Studies Center of National Polytechnic Institute (CINVESTAV of IPN), Mexico City