FIGURE 4.

Tagging RNA by a fill-in reaction with φ29 DNA polymerase. (A) 0.5 µM chemically synthesized, radiolabeled RNA N₂₂ was annealed to 0.75 µM of two different DNA oligonucleotides to form the hybrids indicated. One oligonucleotide (Table 1, “fill in”) was perfectly complementary to the RNA, the other (Table 1, “unpaired fill in”) had a 2-nt mismatch, as indicated in the scheme on top. (Arrow) The position where the incorporation of the biotinylated nucleotide is expected. The fill-in reaction was started by the addition of 7 units/µL φ29 DNA polymerase in the presence of 0.2 mM all four regular dNTPs or with dTTP substituted by 0.5 mM biotin-11-dUTP, as indicated. Samples where taken at the indicated time points, stopped by mixing with 20 mM EDTA in formamide and analyzed by denaturing polyacrylamide gel electrophoresis. DNA marker sizes are indicated on the right. The band labeled with a single dot corresponds to the addition of a nontemplated regular nucleotide; bands labeled with two dots correspond to the addition of a nontemplated biotinylated nucleotide. (B) A DNA polymerase reaction was carried out as in A but with different DNA oligonucleotides. The oligonucleotide shown in the scheme on the left (Table 1, “recessed fill in”) had a 2-nt mismatch to the RNA followed by an additional 2-nt 5′ overhang. The template position directing the incorporation of biotin-dUMP (labeled with an arrow) was “covered” by the mismatch, such that biotin incorporation required 3′ resection of the RNA by the proofreading activity of the DNA polymerase. The oligonucleotide shown on the right (Table 1, “fill in”) had a perfect match to the RNA 3′ end followed by a 4-nt 5′ overhang containing the position directing the incorporation of biotin-11-dUMP (marked with an arrow). DNA marker sizes are indicated on the right. Bands were labeled with one or two dots as in A. (C) A fill-in reaction was carried out as in A with four different DNA oligonucleotides differing in 2 nt at their 5′ ends (from left to right: “fill in,” “fill in” TG, “fill in” GG, “fill in” TT). Reactions were done in the presence of 0.2 mM all four dNTPs, i.e., biotin-11-dUTP was omitted. The DNA marker size is indicated on the left. (D) A fill-in reaction was carried out as in A including substitution of dTTP by biotin-11-dUTP. The concentration of the biotinylated nucleotide was varied as indicated. The regular dNTPs were present at 0.2 mM. The DNA marker sizes are indicated on the right. (E) Fill-in reactions were carried out as in A. An enzymatically synthesized, radiolabeled RNA (0.5 µM) of 200 nt was preincubated with 0.75 μM N₂₀₀ “fill in” at 30°C for the times indicated. Amounts of φ29 DNA polymerase were varied as indicated. Biotin-11-dUTP was used at a concentration of 0.5 mM where indicated. The DNA marker sizes are indicated on the left.