Methods in Anopheles Research - MR4

Chapter 7 : Taxonomy and Systematics7.2 Ribosomal DNA PCR assays7.2.3 General PCR to amplify a portion of the 18S subunit in anophlinesPage 1 of 27.2.3 General PCR to amplify a portion of the 18S rDNA subunit in anophelinesMR4 Vector ActivityIntroductionThe rDNA is a multicopy (>100) locus useful for phylogenetic studies due to its rapid, concerted evolution.It is employed to distinguish between cryptic species since nucleotide differences will quickly fix aftergene flow between two populations ceases.PCR assay for the amplification of the 18S subunit regionPrepare PCR Master Mix for 96, 48 or 1 25μl PCR reactions. Add reagents in the order presented.96 48 1 Reagent1.54 ml 770 μl 15.4 μl Distilled H2O500 μl 250 μl 5.0 μl 5X PCR buffer150 μl 75 μl 1.5 μl dNTP (2mM concentration)100 μl 50 μl 1.0 μl 18SForDros (25pmol/ul) [GAG GGA GCC TGA GAA ACG GCT AC]100 μl 50 μl 1.0 μl 18SRevDros (25pmol/ul) [CCT TCC GTC AAT TCC TTT AAG TTT C]100 μl 50 μl 1.0 μl MgCl2 (25 mM)10 μl 5.0 μl 0.1 μl Taq DNA polymerase (5U/ μl)2.5 ml 1.25 ml 25 μl Total (To each 25 μl reaction add 1.0 μl template DNA)Table 7.2.3.1. F and R indicate forward and reverse orientation. DNA extraction negative control to beincluded in addition to PCR reaction mix negative control.PCR Cycle conditions94°C/5min x 1 cycle(94°C/1min , 54°C/1min , 72°C/1min) x 35 cycles72°C/7min x 1 cycle4°C holdUse a 1.5% Agarose ethidium bromide gel for visualization.Primers create an approximately 900 bp band (Figure 7.2.3.1).Figure 7.2.3.1. Lane 1 1Kb marker, Lane 1, 1kb ladder, lanes 2-9 An.subpictus, lanes 10-11 An. gambiae.