Methods in Anopheles Research - MR4

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Chapter 4 : Stock Authentication4.2 Authentication by PCR4.2.3 Anopheles gambiae white gene v 1Page 1 of 24.2.3 Anopheles gambiae white geneLiz Wilkins, Alice Sutcliffe, Paul HowellIntroductionThe white gene of Anopheles gambiae is located on the X chromosome and known for intron variation,making it a good marker for differentiating closely related species (Mukabayire et al. 2001). In laboratorieswhere several wild stocks from the same sub-species and origin are reared concurrently, it can be difficultto ensure that contamination events between the stocks has not occurred. The MR4 cultures two pairs ofsuch closely related stocks, MOPTI and MALI, ZAN/U and PIMPERENA. An 800 bp segment of intron 3of the white gene ((Besansky et al. 1995); GenBank accession number U29485) was amplified usingprimers WG2 GAGCATCATTTTTTGCTGCG and WG5 CGTGGTTATCGTATCAAAAG as published by(Mukabayire et al. 2001) and sequenced to discover single nucleotide polymorphisms (SNPs) betweenthe stocks (Figure 4.2.3.2.). Primers were designed using the intentional mismatch primer (IMP) method(Wilkins et al. 2006) at these sites for a stock specific authentication method.PCR authentication for stock specific SNP sites in the white genePrepare PCR Master Mix for 96, 48 or 1 25μl PCR reactions. 196 48 1 Reagent1085 μl 542.5 μl 10.85μl Distilled H2O500 μl 250 μl 5.0 μl 5X GoTaq PCR Buffer200 μl 100 μl 2.0 μl dNTP (2.5 mM concentration)100 μl 50 μl 1.0 μl UFOR (25 pmol/ μl) ATT ATC TGA TGA AGC TTG GAG TCT TTT100 μl 50 μl 1.0 μl UREV (25 pmol/ μl) ATG AAA ATA AGG AGC TTC CTG GCA T100 μl 50 μl 1.0 μl PIMPR (25pmol/ μl) TCA ATG ACA TGA CGT TAT AAT CTG TCT TT100 μl 50 μl 1.0 μl MOPR (25pmol/ μl) CTG TTG TCT TAC AGT AGG GTT ATG T100 μl 50 μl 1.0 μl MOP2R (25pmol/ μl) AAC GTA CGA CGT ATG ATC TAA CTG A100 μl 50 μl 1.0 μl MALR (25pmol/ μl) CTC ATA TTC AAG GAT GAA CAC AAT AC100 μl 50 μl 1.0 μl MgCl2 (25 mM)15 μl 7.5 μl 0.15 μl Go-Taq DNA polymerase2.5 ml 1.25 ml 25 μl Total (To each 25 μl reaction add 1 μl template DNA)Table 4.2.3.1. Add reagents in order presented.PCR cycle conditions94°C/5min x 1 cycle(94°C/30sec , 56°C/30sec , 72°C/30sec) x 35 cycles72°C/10min x 1 cycle4°C hold1 Amounts for larger master mixes have been adjusted upwards to be sufficient for 50 and 100 reactionsto compensate for imprecise measurements.
Figure 4.2.3.1 Lane 1 1kb ladder, lane 2Chapter 4 : Stock Authentication4.2 Authentication by PCR4.2.3 Anopheles gambiae white gene v 1Page 2 of 2Run samples on a 2% agarose EtBr gel. (Figure 4.2.3.1).Primers create fragments of 478 universal, 413 mopti, 350 mopti & zan/u,292 mali, 116 pimperenaFigure 4.2.3.2. Specific SNP sites in the white gene used to create this assay. Numbering based onaccession number U29485.SNP sites11591 11767 11826 11885U29485 TTACTATGAC GATTTCTTGT GCTTGTTAGA GACGCTTTACMopti TTACTATGAC GATTTCTTGT GTCTGTTAGA GACACTTAACMaliTTACTATGAC GATGTCTTGT GCTTGTTAGA GACGCTTTACPimperena TTACAATGAC GATTTCTTGT GCTTGTTAGA GACGCTTTACZan/u TTACTATGAC GATTTCTTGT GTCTGTTAGA GACGCTTTACReferencesBesansky NJ, Bedell JA, Mukabayire O, Hilfiker D, Collins FH (1995) The white gene of Anophelesgambiae: structure and sequence of wild type alleles. submittedMukabayire O, Caridi J, Wang X, Toure YT, Coluzzi M, Besansky NJ (2001) Patterns of DNA sequencevariation in chromosomally recognized taxa of Anopheles gambiae: evidence from rDNA and single-copyloci. Insect Mol Biol 10:33-46Wilkins EE, Howell PI, Benedict MQ (2006) IMP PCR primers detect single nucleotide polymorphisms forAnopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin inAnopheles arabiensis. Malar J 5:12596 well PCR sample preparation template
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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