2.4. Mitochondrial lineage determination by mt DNA Cyt b PCR-RFLP
Polymerase chain reaction (PCR) was used to amplify a 550-bp fragment containing the 3’ end of the Cytochrome b mitochondrial gene (mt DNA Cyt b) following the methodology described by Esseghir et al. (1997).
For Restriction fragment length polymorphism (RFLP), digestion of the 550-bp mtDNA Cyt b fragment was carried out with Hae III (Thermo Scientific, Germany). The reaction was performed at 37°C for 10 minutes in a 20 µl total volume, containing 16 µl of PCR product, 2 µl of enzyme (10 U/µl) and 2 µl of standard buffer (10X). The digested samples were separated by electrophoresis in a 3% agarose gel and their sizes determined by comparison with HyperLadder V (Bioline, UK) leading to a characteristic banding pattern for each of the four mitochondrial lineages: Lineage I, two fragments (290 and 220 bp); Lineage II, two fragments (290 and 260 bp); Lineage III, three fragments (290, 140 and 110 bp) and Lineage IV, two fragments (330 and 220 bp) (Merino-Espinosa et al. 2016).