10.2.4. Removal of tagged-cDNA primers from the cDNA reaction
Since the purpose of using tagged-cDNA primers is to amplify only with the tag sequence, it is important to either remove or inactivate the original tagged-cDNA primers after the cDNA reaction, and prior to PCR. If not, then these tagged-cDNA primers (which contain virus-specific sequences) can participate in the PCR reaction, just like a non-tagged virus-specific cDNA primer would. The presence of tagged-cDNA primers in the PCR reaction therefore permits the amplification of false-, self- or misprimed cDNAs of the ‘wrong’ strand, leading to an incorrect conclusion of strand-specificity (Craggs et al., 2001; Peyrefitte et al., 2003; Plaskon et al., 2009; Boncristiani et al., 2009).
Tagged-cDNA primers can be most easily removed from the cDNA reaction using commercial PCR/cDNA purification columns (Peyrefitte et al., 2003). An alternative is to use biotinylated tagged-cDNA primers and then capture the tagged cDNA with Streptavidin-conjugated magnetic beads (Boncristiani et al., 2009). Although highly effective at removing primers, the disadvantage of cDNA purification is that its DNA recovery efficiency of individual columns can be highly variable (Tentcheva et al., 2006), leading to different types of error in (quantitative) detection and interpretation. This can be managed by adding a passive ‘reference’ DNA prior to cDNA purification (similar in concept to the “exogenous internal reference standards” used in RT-qPCR quantification, discussed in the BEEBOOK paper on molecular techniques; Evans et al., 2013), which can be used to normalize the data again afterwards.