Methods in Anopheles Research - MR4

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Chapter 8 : Field Techniques8.4 Species Complex Authentication by PCR8.4.7 Anopheles quadrimaculatus Complex – Cornel et al.Page 1 of 28.4.7 Anopheles quadrimaculatus Species Complex (Cornel et al.)MR4 StaffIntroductionThough malaria is no longer endemic in the United States, the competent vector An. quadrimaculatus isstill present. It has been determined that the An. quadrimaculatus complex is made of at least fivemembers: An. inundatus, An. diluvialis, An. maverlius, An. smaragdinus, and An. quadrimaculatus(Narang et al. 1990). A PCR based on SNPs in the internal transcribed spacer region has beendeveloped for An. quadrimaculatus that can distinguish the various members of the complex (Cornel et al.1996).PCR authentication for the members of the Anopheles quadrimaculatus complex (Cornel et al.1996)Prepare PCR Master Mix for 96, 48 or 1 25 μl PCR reactions. 1 Add reagents in the order presented.96 48 1 Reagent450 μl 225 μl 4.5 μl sterile H 2 O250 μl 125 μl 2.5 μl 10X Perkins Elmer PCR Buffer100 μl 50 μl 1.0 μl dNTP (2 mM mix)200 μl 100 μl 2.0 µl MgCl 2 (25 mM)160 μl 80 μl 1.6 μl AQU (F, 1 pmol/μl) [CGACACAGCTCGATGTACAC]100 μl 50 μl 1.0 μl AQA (R, 1 pmol/μl) [TCCGTAGGAGGCTGCATTTT]460 μl 230 μl 4.6 μl AQB (R, 1 pmol/μl) [CACACTACACAACACGCTTT]230 μl 115 μl 2.3 μl AQC (R, 1 pmol/μl) [TACCCCGGCCTTGTAGCAAA]540 μl 270 μl 5.4 μl AQD (R, 1 pmol/μl) [ATGCAAAAGGTGGTGTTGTG]12.5 μl 6.25 μl 0.125 μl Taq DNA polymerase (5U/μl)2.5 ml 1.25 ml 25 μl Total (To each 24 ul reaction add 1 μl template DNA)Table 8.4.7.1. F and R indicate forward and reverse orientation. Use 1 μl DNA template. If all primers arenot used, complete the volume with water.PCR Cycle conditions94°C/2min x 1 cycle(94°C/1min, 50°C/2min, 72°C/2min) x 25 cycles72°C/10min x 1 cycle4°C holdRun samples on a 2% agarose EtBr gel; load 5 μl samplePrimers create fragments of 319bp An. quadrimaculatus, 227bp An. smaragdinus, 293bp An. inundatusor An. diluvialis, and 141bp An. maverlius.1 Amounts for larger master mixes have been adjusted upwards to be sufficient for 50 and 100 rxnscompensate for imprecise measurements.
Chapter 8 : Field Techniques8.4 Species Complex Authentication by PCR8.4.7 Anopheles quadrimaculatus Complex – Cornel et al.Page 2 of 2ReferencesCornel AJ, Porter CH, Collins FH (1996) Polymerase chain reaction species diagnostic assay forAnopheles quadrimaculatus cryptic species (Diptera: Culicidae) based on ribosomal DNA ITS2sequences. J Med Entomol 33:109-116Narang SK, Seawright JA, Kaiser PE (1990) Evidence for microgeographic genetic subdivisions ofAnopheles quadrimaculatus species C. J Am Mosq Control Assoc 6:179-18796 well sample preparation template
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\Methods inAnopheles ResearchSecond
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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INTRODUCTIONChapter 5 : Insecticide
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Chapter 5 : Insecticide Resistance5
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Chapter 6 : Dissection Techniques6.
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