Methods in Anopheles Research - MR4

Chapter 8 : Field Techniques8.6 Molecular Karyotyping PCR Assays8.6.1 Anopheles gambiae 2La inversion – White et al.Page 1 of 28.6 Molecular Karyotyping PCR Assays8.6.1 Anopheles gambiae 2La inversion (White et al.)Bradley White and Nora BesanskyIntroductionThe 2La inversion in An. gambiae is associated with tolerance to aridity and indoor resting behavior. Thisinversion may also be involved in the incipient speciation between the Mopti and Savanna chromosomalforms (White et al. 2007). Traditionally, polytene chromosomal banding patterns were used to differentiatebetween the 2La and 2L+ a forms. However, due to the complex procedures needed to produce these,few laboratories have trained personnel. In order to better understand the effects of karyotype onphenotype, a PCR-based method was developed based on the published sequence of An. gambiae. Thismethod has been shown to be robust in identifying the inversion within all members of the An. gambiaecomplex.PCR authentication for the 2La inversion in the Anopheles gambiae complex (White et al. 2007)Prepare PCR Master Mix for 96, 48 or 1 25μl PCR reactions. 1 Add reagents in the order presented.96 48 1 Reagent1185 μl 592.5μl 11.85μl Distilled H2O500 μl 250 μl 5.0 μl 5X PCR buffer200 μl 100 μl 2.0 μl dNTP (2.5 mM concentration)100 μl 50 μl 1.0 μl 23A2 (R, 25 pmol/ μl) CTC GAA GGG ACA GCG AAT TA100 μl 50 μl 1.0 μl 27A2 (F, 25 pmol/ μl) ACA CAT GCT CCT TGT GAA CG100 μl 50 μl 1.0 μl DPCross 52L+ (F, 25pmol/ μl) GGT ATT TCT GGT CAC TCT GTT GG200 μl 100 μl 2.0 μl MgCl2 (25 mM)15 μl 7.5 μl 0.15 μl Go-Taq DNA polymerase (5U/ μl)2.4 ml 1.20 ml 24 μl Total (To each 24 μl reaction add 1 μl template DNA)Table 8.6.1.1. F and R indicate forward and reverse orientationPCR cycle conditions94°C/2min x 1 cycle(94°C/30sec , 60°C/30sec , 72°C/45sec) x 35 cycles72°C/10min x 1 cycle4°C holdRun samples on a 2% agarose EtBr gel. (Figure 8.6.1.1).Primers create fragments of 492 and 207bp for 2La and 2L+ a arrangements, respectively.1 Amounts for larger master mixes have been adjusted upwards to be sufficient for 50 and 100 reactionsto compensate for imprecise measurements.