John M. S. Bartlett.pdf - Bio-Nica.info

152 Olmos et al.
Fig. 1. RT-heminested/nested-PCR scheme. Phase I (reverse transcription and first PCR):
RNA target is copied in a cDNA form by the reverse transcriptase that uses the 3′ end external
oligo (Pe1) as the reaction primer. Subsequently, the first PCR is performed using Pe1 and the
5′ end external primer (Pe2) to produce as intermediate product that will act as the target in the
second PCR amplification. Phase II (heminested or nested-PCR): a second PCR using a 5′ end
internal primer (in the example Pi3) combined with one of the external primers (in the example
Pe1; heminested-PCR) or two internal primers, the 3′ end internal primer (Pi1), and the 5′
end internal primer (Pi2; nested PCR). Note that the internal primers could be used for typing
purposes of the larger fragment amplified in the first reaction.