Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.4 Species Complex Authentication by PCR8.4.5 Anopheles funestus Complex – MR4Page 1 of 28.4.5 PCR Discrimination of the Anopheles funestus ComplexLiz Wilkins, MR4 StaffLike the Koekemoer et al.(2002) assay, the MR4 method of Anopheles funestus complex discrimination isbased on species-specific single nucleotide polymorphisms (SNPs) in the second internal transcribedspacer region (ITS2). However, it also incorporates intentional mismatches into the primers (IntentionalMismatch Primers (IMPs)) to increase the specificity (Wilkins et al. 2006).PCR authentication for the members of the Anopheles funestus groupPrepare PCR Master Mix for 96, 48 or 1 25μl PCR reactions. 1 Add reagents in the order presented.96 48 1 Reagent1000 μl 500 μl 10.0 μl sterile H 2 O500 μl 250 μl 5.0 μl Taq 5X PCR Buffer with MgCl 2100 μl 50 μl 1.0 μl dNTP (2 mM mix)200 μl 100 μl 2.0 μl MgCl 2 (25mM)100 μl 50 μl 1.0 μl UV (F, 5 pmol/μl) [CCG ATG CAC ACA TTC TTG AGT GCC TA]100 μl 50 μl 1.0 μl FUN (R, 5 pmol/μl) [CTC GGG CAT CGA TGG GTT AAT CAT G]100 μl 50 μl 1.0 μl VAN (R, 5 pmol/μl) [AAC TCT GTC GAC TTG GTA GCC GAA C]100 μl 50 μl 1.0 μl RIV (R, 5 pmol/μl) [AAT CAG GGT CGA ACG GCT TGC CG]100 μl 50 μl 1.0 μl PAR (R, 5 pmol/μl) [GCC CTG CGG TCC CAA GCT AGA TT]100 μl 50 μl 1.0 μl RIVLIKE (R, 5 pmol/μl) [CTC CCG TGG AGT GGG GGA TC]100 μl 50 μl 1.0 μl LEES (R, 5 pmol/μl) [GAC GGC ATC ATG GCG AGC AGC]10 μl 5 μl 0.1 μl Taq DNA polymerase (5 U/μl) –MR4 uses GoTaq, Promega2.5 ml 1.25 ml 25 μl Total (To each 25 μl reaction add 1 μl template DNA)Table 8.4.5.1. F and R indicate forward and reverse orientation. DNA extraction negative control to beincluded in addition to PCR reaction mix negative control. If all primers are not needed, complete the totalvolume with water.PCR cycle conditions94°C/4min x 1 cycle(94°C/30sec , 58°C/30sec , 72°C/45sec) x 30 cycles72°C/7min x 1 cycle4°C holdRun samples on a 1.5% agarose EtBr gel; load 10 μl sample1 Amounts for larger master mixes have been adjusted upwards to be sufficient for 50 and 100 rxnscompensate for imprecise measurements.