The vaccine preparation that contained CpG ODN was comprised of 10 μg phosphotioated CpG ODN (5′-TCG TCG TCG TTC GAA CGA CGT TGA T) also known as ODN-M362 (Alpha DNA, Canada) as described previously [ 22].
Briefly, primers (Alpha DNA, Canada) and TaqMan probes (Applied Biosystems, USA) were designed to span exon-intron junctions to prevent amplification of genomic DNA and to result in amplicons of fewer than 150 base pairs to enhance the efficiency of PCR amplification.
An image of an electrocardiogram alludes to the 1950s invention of the pacemaker, and a twist of DNA represents Canada's role in sequencing the human genome.
After purification using the EZ-10 Spin Column DNA Gel Extraction Kit (BBI, Canada), the DNA was inserted into vector pMD18-T following the manufacturer's instructions (TaKaRa, Japan).
Following written informed consent, participants were asked to provide a buccal (using a cytobrush) or saliva sample (Oragene DNA Self-Collection Kit, DNA Genotek Inc., Ottawa, ON Canada) for DNA isolation and to complete repository specific questionnaires.
PCR chemistry for all reactions included 25 ng DNA, 5 μL 2× Qiagen Multiplex mix (Mississauga, ON, Canada), 0.5 μL Ultrapur water (Gibco), and 2 μL primer mix.
The researchers--led by Philip Griebel of the University of Saskatchewan in Saskatoon, Canada--gave four fetal lambs a DNA vaccine for bovine herpesvirus 1.
Three ante mortem sampling and detection methods were chosen for comparison: rectal swabs (CultureSwab Liquid Stuart, BD Canada, Mississauga, ON) for culture on selective agar, qPCR on DNA extracted from fecal samples (200 mg samples, QIAmp DNA stool mini kit, Qiagen Inc ., and qPCR on DNA extracted from GenoTube swabs.
DNA was quantified using a NanoDrop 1000™ (Thermo Fisher Scientific, Ottawa, ON, Canada) and diluted to 1 ng/μL for mt control region sequencing and 10 ng/μL for microsatellite analysis.
Slides were prehybridized with DIG buffer (Roche Diagnostics, Laval, QC, Canada) supplemented with yeast tRNA (4 mg/ml, Invitrogen) and Cot-1 DNA (1 mg/ml, Invitrogen) for 1 hour at 37°C.
Following centrifugation (20 min at 12,000 × g), the clarified lysate was used to isolate chromosomal DNA using the Qiagen® Genomic-tip 500/G genomic DNA isolation kit (Qiagen Inc.-Canada, Ontario, CA).
