Methods in Anopheles Research - MR4

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Chapter 3 : Specific Anopheles Techniques3.7 Anopheles Mating3.7.4 An. gambiae and arabiensis Mating Status DeterminationPage 1 of 43.7.4 An. gambiae and arabiensis Mating Status Determination in PreservedFemalesK.R. Ng’habi and Greg LanzaroIntroductionThe An. gambiae mosquito has a karyotype consisting of two pairs of autosomes (Chromosome 2 and 3)and one pair of sex chromosomes (Chromosome X and Y). The Y chromosome constitutes ~10% of thewhole genome and contains a male determining factor which, when present in a XX/XY system, inducesmale development (Clements 1992). Y-chromosome linked DNA fragments have been characterized andY-chromosome specific PCR markers have been developed (Krzywinski et al. 2004; Krzywinski et al.2005). Previously, detection of mating success among females relied on microscopic dissection of femaleovaries or examination of sperm in the female spermatheca. This method is reliable and robust but withthe limitations that it is time consuming, labor intensive, and requires fresh specimens. A simple andrapid method to determine the mating status of dried female An. gambiae is therefore useful in order toanalyze large sample sizes within a short period of time.Prepare PCR mixture for 96, 48 or 1 50µl PCR reactions 1 . Add reagents in the order presented.96 48 1 Reagent3.73 ml 1.87 ml 38.8 µl Sterile water18.63 ml 9.31 ml 5 µl Taq 10X PCR Buffer with MgCl 2 (1.5 mmol/L)3.73 ml 1.87 ml 0.2 µl dNTP (2.5 mmol/L)931.2 µl 465.6 µl 0.25 µl S23 (F, 25pmol) [CAAAACGACAGCAGTTCC]232.8 µl 116.4 µl 0.25 µl S23 (R, 25pmol ) [TAAACCAAGTCCGTCGCT]116.4 µl 58.2 µl 0.5 µl Taq polymerase27.354 ml 13.677 ml 45 µl Total (To each 45µl add 5µl of DNA template)Table 3.7.4.1. F and R indicate forward and reverse primers, respectively. DNA extractions of males mayalso be used as positive controls.96 48 1 Reagent3.73 ml 1.87 ml 38.8 µl Sterile water18.63 ml 9.31 ul 5 µl Taq 10X PCR Buffer with MgCl 2 (1.5 mmol/L)3.73 ml 1.87 ml 0.2 µl dNTP (2.5 mmol/L)931.2 µl 465.6 µl 0.25 µl 128125I (F, 25pmol) [GGCCTTAACTAGTCGGGTAT]232.8 µl 116.4 µl 0.25 µl 128125I (R, 25pmol) [TGCTTTCCATGGTAGTTTTT]116.4 µl 58.2 µl 0.5 µl Taq polymerase27.354 ml 13.677 ml 45 µl Total (To each 45µl add 5µl of DNA template)Table 3.7.4.2. F and R indicate forward and reverse primers, respectively. DNA extractions of males mayalso be used as positive controls.PCR cycle conditions94 o C/3min x I cycle(94 o C/20sec, 55 o C-64 o C/30sec and 72 o C/1 min)* x 35 cycles72 o C/10min x 1 cycle4°C hold1 Amounts for larger master mixes have been adjusted upwards to be sufficient for 50 and 100 rxnscompensate for imprecise measurements.
Chapter 3 : Specific Anopheles Techniques3.7 Anopheles Mating3.7.4 An. gambiae and arabiensis Mating Status DeterminationPage 2 of 4Run samples on a 3.0 % agarose EtBr gel for visualization. *The primers have different amplificationtemperatures but within this range should work.Figure 3.7.4.1: Agarose gel electrophoresis showing amplification of Y chromosome sequences in An.arabiensis (primer 128125I) males and mated females. There was no amplification in virgin females.Lanes 1 and 26, ladder, lanes 2-13 mated, lanes 14-19 unmated, lanes 20-25 males. (Ng'habi et al.2007), used with permission.Figure 3.7.4.2: Agarose gel electrophoresis showing amplification of Y chromosome sequences in An.gambiae s.s. (Primer S23) males and mated females. There was no amplification in virgin females. Lanes1 and 26, ladder, lanes 2-13 mated, lanes 14-19 unmated, lanes 20-25 males. (Ng'habi et al. 2007), usedwith permission.
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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