How are the primers validated?
Each primer pair undergoes extensive in silico and in chemico validations ensuring specificity and high sensitivity. Primer design parameters were selected to enhance: • Organism specificity with minimized SNP interference (where possible) • Gene specificity • Amplicon efficiency • Uniformity of primer Tm’s • A/T-rich 3′-ends of primers • Elimination of primer-dimer artifacts • Generation of single-peak dissociation curves • Detection of single bands of expected size via gel electrophoresis The process begins by running a constant amount of gDNA template per well in quadruplicate reactions using a hot-start based SYBR® Green I detection system. The reactions are run using Applied Biosystems 7900HT FAST Real-Time PCR System default conditions followed by acquisition of a standard dissociation curve. The first round of evaluation begins with the elimination of any primer pair that generates a multi-peak dissociation curve (DC test). Primer pairs that pass the DC test are evaluated by the
