Figure 8
Digestion of fluorescently-labeled annealed oligonucleotide substrates by lambda-Exo. In experiments analogous to those described for SXT-Exo (see Figure 7), the ability of lambda-Exo to digest three different (partially) dsDNA substrates was investigated. In each assay, lambda-Exo (3 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 5 mM MgCl2. Aliquots were removed and quenched at 0, 0.5, 1, 2, 4 and 10 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 by SXT-Exo. Panel B: Representative gel image showing digestion of the Blunt ended DNA substrate by lambda-Exo. Panel C: Representative gel image showing digestion of the 3'-overhang DNA substrate by lambda-Exo. Panel D: Plot showing the digestion of the three DNA substrates by lambda-Exo over a 10 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.