10.2.5. Inactivating tagged-cDNA primers in the cDNA reaction
It is also possible to inactivate the tagged-cDNA primer in the cDNA reaction, and so prevent it from participating in the PCR reaction. This can be done using exonuclease-I, which specifically digests only single-stranded DNA, i.e. the tagged-cDNA primer (Craggs et al., 2001; Purcell et al., 2006; Plaskon et al., 2009; Lin et al., 2009) or by phosphorylating the 3’ end of the tagged-cDNA primer (making it impossible for the polymerases in PCR to synthesize DNA from this primer). These enzymatic reactions can be done right after the cDNA reaction, in the same reaction tube, after which the enzymes can be heat-inactivated prior to the PCR reaction. The exonuclease-I digestion has become the method of choice for quantitative strand-specific RT-qPCR (e.g. Purcell et al., 2006; Plaskon et al., 2009; Lin et al., 2009; Runckel et al., 2011). The main advantages of enzymatic inactivation of the tagged-cDNA primers, compared to the primer removal methods (see section 10.2.4.), is that enzymatic inactivation is much faster and cheaper, with fewer handling and contamination errors (no tube changes), and that it avoids the possible quantitation errors of the primer removal methods.