Methods in Anopheles Research - MR4

Chapter 7 : Taxonomy and Systematics7.2 Ribosomal DNA PCR Assays7.2.1 Amplification of the Second Internal Transcribed Spacer Region (ITS2) in AnophelinesPage 1 of 27.2 Ribosomal DNA PCR Assays7.2.1 Amplification of the Second Internal Transcribed Spacer Region (ITS2)in AnophelinesMR4 StaffIntroductionIn many insect genera, often the amplification and sequencing of the ITS2 region is useful in order todetect intraspecific differences. The ribosomal DNA region is made up of 3 functional rDNA genesseparated by two spacer regions: the ITS1 and ITS2 regions. In many anopheline species it has beenfound that the ITS2 region is more informative, especially in designing PCR assays to distinguishmembers of cryptic complexes. Universal primers designed by Beebe and Saul (1995) and have beenshown to be suitable in a number of anopheline species.PCR assay for amplifying the ITS2 region in anopheline mosquitoes (Beebe and Saul 1995)Prepare PCR Master Mix for 96, 48 or 1 25 μl 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)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]30 μl 15 μl 0.3 µl MgCl 2 (25 mM)15 μl 7.5 μl 0.15 μl Go-Taq DNA polymerase (5 U/μl)2.5 ml 1.25 ml 24 μl Total (To each 24 ul reaction add 1 μl template DNA)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 holdPCRRun samples on a 2.0% agarose EtBr gel for visualization. Band sizes will vary by species (Figure7.2.1.1).ReferencesBeebe NW, Saul A (1995) Discrimination of all members of the Anopheles punctaulatus complex bypolymerase chain reaction-restriction fragment length polymorphism analysis. Am J Trop Med Hyg53:478-4811 Amounts for larger master mixes have been adjusted upwards to be sufficient for 50 and 100 rxnscompensate for imprecise measurements.