Excitingly, primer extension was observed beyond the first stop site, generating the second ("2*") and third ("3*") stops sites (Figure 2A and 2B).
Indeed, HIV-1 RT displayed much more extension beyond the first stop site ("1*": see the sites marked with "U" in Figure 2D) and full length products (see "]" in Fig. 2D) than MuLV RT (Fig. 2E).
The efficiency of the primer extension beyond the first stop site in the total primer extension has been used to compare the enzyme fidelity between polymerases [5], [22], [25].
Also, HIV-1 RT showed much higher extension beyond the first stop site (Fig. 2D) than MuLV RT (Fig. 2E), suggesting that HIV-1 RT has lower fidelity than MuLV RT, which has been demonstrated previously (25).
Now with the misincorporation assays completed with the three other polymerases, we were able to compare their enzyme fidelity by comparing the extent of the primer extension beyond the first stop site (see "*" in Fig. 2).
Interestingly, IAV Pol complex generated similar or slightly less primer extension beyond the first stop site than the high fidelity MuLV RT even if a lower polymerase activity of MuLV RT, compared to IAV Pol, was used, supporting that IAV Pol complex has at least similar fidelity with MuLV RT.
