Taqgold

IgH and IgK rearrangements were studied by multiplex PCR with BIOMED-2 primers using a BIOMED-2 PCR based protocol [22 (link)]. The final 50-μl reaction volume consists of 40-μl mastermix, 1-2U of TaqGold (Applied Biosystems) and 100 ng of DNA for both IgH and IgK reactions. The PCR reactions were performed on a ABI 9700 thermal cycler with the following amplification parameters; initial denaturation for 15 minutes at 94°C, followed by 32 cycli of 30 seconds at 95°C, 30 seconds at 60°C and 30 seconds at 72°C, with a final extension step of 10 minutes at 72°C. PCR product was added into the appropriate well of a 96-well PCR plate. A mix of HiDi Formamide and GeneScan-500 LIZ Size Standard was added into each well and mixed with 2 μl PCR product and finally loaded to the ABI3130xl analyzer. Paraffin sections from representative mouse lesions and matched frozen original tumor biopsies were used to isolate genomic DNA. The quality of the extracted DNA was excellent, showing an amplification up to 400 bp.

Each qPCR reaction contained 5 μl of pre-amplified DNA (diluted 1:10 with diethylpyrocarbonate-treated water), Core Buffer, 3 mM Mg, 0.35 pmoles ROX reference Dye, 2 U TaqGold (N8080247, Applied Biosystems), 16 nmoles dNTPs (N0447L, New England BioLabs, Ipswich, MA, USA), 10 pmoles forward and reverse primers (PM607 and PM585, Integrated DNA Technologies, Skokie, IL, USA), 10 pmoles probe, 5.2 mU RNase H2 enzyme (11-02-12-01, Integrated DNA Technologies, Skokie, IL, USA), Exogenous IPC Mix, and Exogenous IPC DNA for a total volume of 20 μl. qPCR was performed on an automated fluorometer (ABI PRISM 7900 HTA FAST, Thermo Fisher Scientific). The following amplification conditions were used: 9 min at 95°C, 50 cycles of 30 s at 95°C and 1 min at 63°C. Fluorescent signals were collected during the annealing phase and Cq values extracted at adjusted baselines and thresholds appropriate for FAM (watersnake assay) and VIC (internal positive control) fluorophores.

We extracted DNA from 14 adult Ae. albopictus collected in Florida (FL1 and FL2) using DNAeasy tissue kits (Qiagen, Valencia, California, USA). We chose 6 survivors and 6 dead specimens following DDT exposure and amplified portions of domains II, III, and IV of the voltage-gated sodium channel (VGSC), a known target of DDT and pyrethroid insecticides, using primers from Kasai et al. [43] (link). Specifically we amplified and sequenced domain II with aegSCF20 and aegSCR21, domain II with aegSCF7 and aegSCR8, and domain IV with albSCF6 and albSCR8. Our PCR was composed of 1× PCR buffer, 2.5 mM of MgCl₂ (2.0 mM for Domain III), 200 µM of each dNTP, 0.2 mg/ml of BSA, 0.2 µM of each primer, and 1 unit of TaqGold (Applied Biosystems, Foster City, California, USA). The PCR cycle started with a 10 min denaturation (and TaqGold activation) at 96°C followed by 40 cycles of 30 s at 96°C, 30 s at 55°C (Domain II and IV) or 53°C (Domain III) and 45 s at 72°C, and a final extension of 10 min at 72°C. The PCR products were cleaned with ExoSAP-IT (USB, Cleveland, Ohio, USA) and cycle sequenced for analyses on an ABI 3100 automated sequencer (Applied Biosystems). Sequences were cleaned and checked with Sequencher 5.0 (Gene Codes, Ann Harbor, Michigan, USA).