Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.5 Real-Time PCR Assays8.5.1 Vector Population Monitoring Tool using Real-Time PCR8.5.1.4 Insensitive acetylcholinesterase (iAChe) assayPage 1 of 28.5.1.4 Insensitive acetylcholinesterase (iAChe) assayChris Bass, Martin Williamson, John Vontas, Hilary Ranson, Martin Donnelly and Lin FieldIntroductionThis assay detects the G119S mutation in the gene ace-1 which encodes the acetylchoinesteraseenzyme. This assay is suitable for use on real-time PCR machines that have two or more detectionchannels. A PCR based assay can be found in Chapter 5.3.4.Assay conditions96 48 1 Reagent500 μl 250 μl 5.0 μl sterile H 2 O1.0 ml 500 μl 10.0 μl SensiMix DNA kit (Quantace)160 μl 80 μl 1.6 μl primer ACE1-F (800nM) GGCCGTCATGCTGTGGAT160 μl 80 μl 1.6 μl primer ACE1-R (800nM) GCGGTGCCGGAGTAGA40 μl 20 μl 0.4 µl TaqMan MGB probe (200 nM) Ace1G119 VIC-TTCGGCGGCGGCT40 μl 20 μl 0.4 µl TaqMan MGB probe (200 nM) Ace1S119 6FAM-TTCGGCGGCAGCT1.9 ml 950 μl 19 μl Total (To each 19 μl reaction add 1-2 μl genomic DNA)Assay PCR cycle conditions95°C/10 min x 1 cycle; (95°C/10sec, 60°C/35sec) x 40 cyclesMeasure fluorescence at the end of each cycleInterpreting Results and ExamplesThe iAChE assay uses two probes, the first specific for the wild-type allele is labeled with VIC and thesecond, specific for the mutant allele (S119), is labeled with FAM. A substantial increase in VICfluorescence indicates a homozygous wild-type, a substantial increase in FAM fluorescence indicates ahomozygous mutant and a, usually intermediate, increase in both signals indicates a heterozygote(Figure 8.5.1.4.1).