Methods in Anopheles Research - MR4

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Chapter 7 : Taxonomy and Systematics7.3 Mitochondrial DNA PCR assays7.3.2 General PCR to amplify the cytochrome B region in anophlinesPage 1 of 27.3.2 General PCR to amplify the cytochrome B region in anophelinesMR4 Vector ActivityIntroductionAlthough most phylogenetic studies involve one of the cytochrome oxidase genes (COI and COII) or oneof the D domains in the 28S subunit of the rDNA, the cytochrome B (cytb) gene has been usedextensively by vertebrate scientists (Simmons 2001). The utility of mitochondrial DNA (mtDNA) forphylogenetic studies is based on its lack of recombination, maternal inheritance, rapid evolution, andintraspecific polymorphisms (Avise et al. 1987).PCR assay for the amplification of the cytochrome B regionPrepare PCR Master Mix for 96, 48 or 1 50μl PCR reactions. Add reagents in the order presented.96 48 1 Reagent3.32 ml 1.66 ml 33.2 μl sterile H 2 O1.0 ml 500 μl 10.0 μl 5X PCR Buffer200 μl 100 μl 2.0 μl dNTP (2 mM mix)200 μl 100 μl 2.0 μl cytbF (25 pmol/ μl) GGA CAA ATA TCA TTT TGA GGA GCA ACA G200 μl 100 μl 2.0 μl cytbR (25pmol/ μl) ATT ACT CCT CCT AGC TTA TTA GGA ATT G60 μl 30 μl 0.6 μl MgCl 2 (25mM)20 μl 10 μl 0.2 μl GoTaq DNA polymerase (5U/μl)5.0 ml 2.5 ml 50 μl Total (To each reaction add 1.0 µl of DNA)Table 7.3.2.1. F and R indicate forward and reverse orientation.PCR Cycle conditions94°C/5min x 1 cycle(94°C/30s , 50°C/30s , 72°C/1min) x 35 cycles72°C/10min x 1 cycle4°C holdUse a 1.5% Agarose ethidium bromide gel for visualization.Primers create an approximately 459 bp band (Figure 7.3.2.1).Figure 7.3.2.1. Lane 1 1Kb marker, lane 2 An. atroparvus, lane 3 An.gambiae, lane 4 An. arabiensis, lane 5 An. funestus, lane 6 An.pharoensis.
Chapter 7 : Taxonomy and Systematics7.3 Mitochondrial DNA PCR assays7.3.2 General PCR to amplify the cytochrome B region in anophlinesPage 2 of 296 well PCR sample preparation templateReferencesAvise JC, J Arnold, RM Ball, E Bermingham, T Lamb, JE Neigel, CA Reeb, and NC Saunders. 1987.Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics andsystematics. Ann Rev Ecol Syst. 18: 489-522.Krzywinski, J, RC Wilkerson, and NJ Besansky. 2001. Evolution of mitochondrial and ribosomal genesequences in Anophelinae (Diptera: Culicidae): Implications for phylogeny reconstruction. Mol Phyl Evol.18 (3): 479-487.Simmons RB and SJ Weller. 2001. Utility and evolution of cytochrome b in insects. Mol Phyl Evol. 20 (2):196-210.
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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Chapter 9 : Obtaining Live Material
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