Lasergene 10

The nucleotide sequence for tick isoatp4056 gene from the genomic region (GenBank acc. no. DS922985) and partial transcript sequence (GenBank acc. no. XM_002414056.1) was downloaded from NCBI and was used to predict the possible 3’ UTR region using Drosophila melanogaster oatp sequence as a reference gene. Using a set of oligonucleotides (as mentioned in S1 Table), isoatp4056 complete CDS along with 3’UTR region was amplified from cDNA prepared from adult female tick RNA. The PCR amplified tick isoatp4056 full-length gene fragment was cloned into pGEM-T-easy vector (Promega, USA) for sequencing and storage. Nucleotide sequencing was performed at Eurofins Genomics (Eurofins, USA) and analyzed using DNASTAR Lasergene 10 (DNASTAR, USA).

The nucleotide variability around the 264-psbA, 2041-ACCase and 106-EPSPS codons was determined for A. retroflexus, L. multiflorum and E. indica respectively in order to design robust and widely applicable PCR primers across different populations and plants and to validate the results generated with the dPACS assays. Eight individual plants from each of the six diverse populations were analysed (Table 1). The PCR primers used for amplifying a DNA fragment around the three different codons are summarised in Table 3. PCR was carried out on the extracted DNA using PuReTaq Ready-To-Go PCR beads (Amersham Biosciences, Bucks, UK) in a total volume of 25 µL containing 0.8 µM of each primer and about 50 ng of genomic DNA. The reactions were performed on a Master Cycle Gradient Thermocycler Model 96 (Eppendorf, Stevenage, UK) set with a denaturation step at 95 °C for 4 min followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C and 1 min at 72 °C. A final extension step for 10 min at 72 °C was also included. Direct Sanger sequencing (Genewiz LLC, South Plainfield, NJ, USA) was subsequently carried out on 1 μL of neat PCR product using the forward PCR primer. The 48 individual sequences (8 plants × 6 populations) per gene fragment were aligned and compared using the Seqman software (version, DNASTAR Lasergene 10, DNASTAR, Madison, WI, USA).

RNA was extracted from 1 ml plasma aliquots with QIAamp UltraSens Virus RNA kit (Qiagen, Valencia, CA) and reverse-transcribed using SuperScript III First-strand Supermix and random hexamer primer (Invitrogen, Carlsbad, CA). DNA was extracted from LN cell suspensions with QIAamp Blood and Tissue kit (Qiagen). PCR amplification was carried out to amplify the pol gene encoding the first ∼250 amino acids of RT with primers as previously described [15] (link). PCR products were purified with QIAquick gel extraction or QIAquick PCR purification kits (Qiagen). Clones were prepared by Zero-Blunt TOPO cloning (Invitrogen) and transformation of Top10 E. coli (Invitrogen). Individual bacterial colonies were grown in LB-broth (Invitrogen), and DNA was extracted with QIAprep Spin Miniprep kits (Qiagen). The presence of the RT gene was verified by restriction digestion and visualization on a 1% agarose gel, and positive clones were sent for sequencing (GeneWiz, South Plainfield, NJ) with primers specific to HIV_HxB2 RT. Gene sequences were analyzed by Lasergene 10 (DNAStar, Madison, WI). Mutations were classified as NNRTI DRMs as defined by the Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu/) [30] (link).