FIGURE 6.

Typhoon analysis of the products of the RT-FeDEx reaction released by the 5′–3′ exoribonuclease J1 or the 3′–5′ exoribonuclease RNase PH. Separation on 20% polyacrylamide gel (5 M urea). The gel was scanned on a Typhoon to detect the fluorescence associated with FAM. RNA1/DNA1 duplexes are incubated in the presence of Xrn1 or RNase J1, RNA2/DNA2 duplexes in the presence of RNase PH. RNA1 and RNA2 are principally digested to the size of short oligonucleotides linked to FAM. Analysis of an overexposed gel (only lane in the presence of Xrn1 [20 min] is shown on the right) suggest that RNase J1 (J1) and RNase PH (PH) produce short oligonucleotides of 2–3 nucleotides (nt) and 4 or 5 nt, respectively, if one considers that the fast migrating band produced by Xrn1 is a single nucleotide linked to FAM. In the particular case of the RNase PH digestion, a slow migrating band, about 17 nucleotides in length, is likely to correspond to the 5′ proximal sequence of RNA2, protected by base pairings with DNA2 during RNase PH digestion. RNA and DNA concentrations are 0.5 μM and 1 μM, respectively; enzyme concentrations are 50 nM.