Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.4 Species Complex Authentication by PCR8.4.6 Anopheles minimus ComplexPage 1 of 28.4.6 Anopheles minimus Species ComplexMR4 StaffIntroductionAnopheles minimus is a complex comprised of three members: species A (minimus sensu stricto), C(harrisoni), and E. Anopheles minimus and An. harrisoni occur sympatrically throughout Southeast Asiaand are implicated in malaria transmission while species E is limited to the Ryuku Islands of Japan(Garros et al. 2006). An original method for distinguishing the various members was determining thepresence of a pale humeral spot; however this method has proven unreliable in use (Sungvornyothin etal. 2006). A PCR-RFLP (restriction fragment length polymorphism) designed utilizing SNP differences inthe ITS2 regions has been developed to distinguish complex members of An. minimus (Van Bortel et al.2000).PCR authentication for the members of the Anopheles minimus complex (Van Bortel et al. 2000)Prepare PCR Master Mix for 96, 48 or 1 25μl PCR reactions. 1 Add reagents in the order presented.96 48 1 Reagent1555 μl 777.5 μl 15.55 μl sterile H 2 O500 μl 250 μl 5.0 μl GoTaq 5X PCR Buffer with MgCl 2100 μl 50 μl 1.0 μl dNTP (2.5 mM mix)30 μl 15 μl 0.3 µl MgCl 2 (25 mM)150 μl 75 μl 1.5 µl ITS2 A primer (1 pmol/μl) [TGT GAA CTG CAG GAC ACA T]150 μl 75 μl 1.5 µl ITS2 B primer (1 pmol/μl) [TAT GCT TAA ATT CAG GGG GT]15 μl 7.5 μl 0.15 μl Go-Taq DNA polymerase (5 U/μl)2.5 ml 1.25 ml 25 μl Total (To each 24 ul reaction add 1 μl template DNA)Table 8.4.6.1. Prepare PCR Master Mix for 96, 48 or 1 25 μl PCR reactions. Add reagents in the orderpresented. Use 1 μl DNA template.PCR cycle conditions94°C/4min x 1 cycle(94°C/30sec, 53°C/40sec, 72°C/30sec) x 35 cycles72°C/10min x 1 cycle4°C holdRFLP ProcedurePer reaction well:2 μl sterile H 2 O2 μl Restriction Enzyme buffer1 μl Sau 96I restriction enzyme (GGNCC cutting site)15 μl PCR product20 μl Total1 Amounts for larger master mixes have been adjusted upwards to be sufficient for 50 and 100 rxnscompensate for imprecise measurements.