Methods in Anopheles Research - MR4

Chapter 7 : Taxonomy and Systematics7.3 Mitochondrial DNA PCR assays7.3.1 General PCR for amplification of Cytochrome c Oxidase I and II in anophelinesPage 1 of 27.3 Mitochondrial DNA PCR Assays7.3.1 General PCR for amplification of Cytochrome c Oxidase I and II inanophelinesIntroductionMitochondrial DNA (mtDNA) is one of the most commonly studied regions in insect systematics due to itshigh rate of homoplasmy (Caterino et al 2000). Within the mtDNA there are several segments of whichonly a few are routinely examined: cytochrome c oxidase I (COI), cytochrome c oxidase II (COII), andcytochrome b (cytb – see Chapter 7.3.2).Primer sequenceReferenceCOI forward GGA GGA TTT GGA AAT TGA TTA GTT CC Beard 1993COI reverse GCT AAT CAT CTA AAA ATT TTA ATT CCCOII forward TCT AAT ATG GGA GAT TAG TGC Simon 1994COII reverse ACT TGC TTT CAG TCA TCT AAT GTable 7.3.1.1. Primer sequences and references used in these assays.Prepare PCR Master Mix for 96*, 48* or 1 25μl PCR reactions. Add reagents in the order presented.96 48 1 Reagent1.24 ml 620 μl 12.40 μl sterile H 2 O500 μl 250 μl 5.0 μl 5X PCR Buffer (go taq Promega)200 μl 100 μl 2.0 μl dNTP (25 mM mix)150 μl 75 μl 1.5 μl Forward primer (600 nM/ μl)150 μl 75 μl 1.5 μl Reverse primer (600 nM /μl)250 μl 125 μl 2.5 μl MgCl 2 (25mM)10.0 μl 5.0 μl 0.1 μl Taq DNA polymerase (5U/μl)2.5 ml 1.25 ml 25 μl TotalTable 7.3.1.2. Add 1 µl of template gDNA to each reaction. DNA extraction negative control to beincluded in addition to PCR reaction mix negative control.PCR Cycle conditions95°C/3min x 1 cycle(95°C/30sec , 55°C/30sec , 72°C/45sec) x 35 cycles72°C/10min x 1 cycle4°C holdRun samples on a 1.5% agarose gel. You will expect the following fragment sizes: COI – 700bp, COII –730 bp (Figure 7.3.1.1.).