Taq dna polymerase

For confirming and sequencing grgA alleles in the chromosome and plasmid, total DNA was extracted from ~1,000 infected cells using the Quick-gDNA MiniPrep kit (Sigma Millipore) following manufacturer’s instructions. The resulting DNA was used as template for PCR amplification using Taq DNA polymerase (Genscript). DNA fragments resolved with electrophoresis of 1.2% agarose gel purified using the Gel Extraction Kit (Qiagen) and subject to Sanger sequencing at Quintara Biosciences.

Total RNA was extracted using the RNeasy plus mini kit (Qiagen). 200 ng of total RNA were reverse transcribed in a total volume of 20 μl using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. The CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad) was used for real-time RT-PCR. Primers and dual-labeled probes were obtained from Eurofins MWG Operon. Primer and probe sequences were described previously (Hoppstädter et al., 2010 (link)). Standards, from 60 to 0.00006 attomoles of the PCR product cloned into pGEMTeasy (Promega), were run alongside the samples to generate a standard curve. All samples and standards were analyzed in triplicate. The PCR reaction mix consisted of 10x PCR buffer (GenScript), either 2 or 8 mM dNTPs (GenScript), 3–9 mM Mg²⁺, 500 nM sense, and antisense primers, either 2.5 or 1.5 pmol of the respective dual-labeled probe, and 2.5 U of Taq DNA Polymerase (GenScript) in a total volume of 25 μl as described in (Hoppstädter et al., 2010 (link), 2012 (link); Hahn et al., 2014 (link)). The reaction conditions were 95°C for 8 min followed by 40 cycles of 15 s at 95°C, 15 s at a reaction dependent temperature varying from 57 to 60°C, and 15 s at 72°C.

Genomic DNA was extracted from tail clips using the Maxwell 16 System (Promega, Madison, WI, USA). PCR was performed with Taq DNA polymerase (GenScript, Piscataway, NJ, USA) and specific primer sets (Supplementary Table 2) to detect the presence of the transgenes. For Tmc1^∆ genotype analysis, we designed amplification primers specific to the Tmc1 wild-type allele or the Tmc1^∆ allele (Supplementary Table 3). These primers do not recognize the modified BACs Tg[P_Tmc1::Tmc2], Tg[P_Tmc1::Tmc1] or Tg[Tmc1^∆Ex8_9].

Virus detection was done by RT-PCR using total RNA as described by Halgren et al. [11 (link)]. RT was performed using random hexamers and RevertAid^® reverse transcriptase (Thermo Scientific, USA) following manufacturer’s instructions. PCR was carried out in a 10 μL mixture containing 1 μL 10× buffer, 0.2 uL of 10 mM dNTPs, 1.5 μL cDNA template, 6.8 uL of molecular biology grade water, 0.1 μL of Taq DNA polymerase (GenScript, USA) and 0.2 μL of each primer (40 μM) designed and selected based on specificity across closest relatives. PCR parameters were as follows: 94°C for 4 min, 40 cycles of 94°C for 45 s, 57°C for 30 s and 72°C for 45 s, and a final extension step of 10 min at 72°C.
In addition, a commercial ELISA kit (Agdia, USA) developed for the detection of PapMV and AltMV was used to test for serological compatibility.

For validation of the variants found by using WGS data, purified PCR products underwent Sanger sequencing by using Taq DNA Polymerase (Genscript, Piscataway, NJ, USA), a BigDye Terminator v3.1 Cycle Sequencing Kit, and an ABI 3100 or 3500 Genetic Analyzer (Thermo Fisher Scientific).