Figure 7
Digestion of fluorescently-labeled annealed oligonucleotide substrates by SXT-Exo. The ability of SXT-Exo to digest three different (partially) dsDNA substrates (prepared from two annealed oligonucleotides, see Additional File 4) was monitored by quantifying the digestion of the 5'-phosphorylated-3'-Cy3-labeled strand using fluorescence gel scanning. In each assay, SXT-Exo (50 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 0.5 mM MnCl2. Aliquots were removed and quenched at 0, 1, 2, 4, 10 and 20 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A: Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate (annealed 5'-PO4-70Cy3 + 50blunt oligonucleotides) by SXT-Exo. Panel B: Representative gel image showing digestion of the Blunt ended DNA substrate (annealed 5'-PO4-50Cy3 + 50blunt oligonucleotides) by SXT-Exo. Panel C: Representative gel image showing digestion of the 3'-overhang DNA substrate (annealed 5'-PO4-50Cy3 + 70overhang oligonucleotides) by SXT-Exo. Panel D: Plot showing the digestion of the three DNA substrates by SXT-Exo over a 20 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.