FIG. 6.

Blocking primer prevents self-priming of −sssDNA and promotes specific strand transfer. The 100-base −sssDNA forms a hairpin similar to the TAR RNA hairpin, which can self-prime and be extended by RT. NC promotes the annealing of a blocking primer 17 bases or longer to the 3′ end of the −sssDNA, preventing self-priming. The blocking primer is released in the presence of NC only if it is displaced by an acceptor which has greater complementarity to the −sssDNA. During reverse transcription, the blocking primer(s) would be a fragment or fragments of the RNA genome that remained annealed to the −sssDNA after synthesis was complete.