Editing Purification of Tth-Muts (section)

== Mismatch DNA binding assay ==

Mismatch DNA binding was measured by gel retardation assay. Complementary strands of a 16mer or a 37mer oligonucleotide were synthesized (Fig. 6). Each top strand was labeled at the 5′ end with [γ-32P]ATP using with polynucleotide kinase. The oligonucleotides were annealed at 70°C (16mer) or 95°C (37mer) for 10 min, allowed to cool to 30°C (at 1°C/min), and then placed on ice until required. When the assay was carried out using 37 bp duplex DNA as a substrate, each binding assay reaction mixture (total volume 10µl), comprising 0.2 pmol oligonucleotide duplex, the assay buffer [50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 5 mM β-mercaptoethanol, 10% (v/v) glycerol and 20 mM MgCl2] and T.thermophilus MutS protein, was incubated at 35 or 60°C for 30 min. After adding 2 µl 50% (w/v) sucrose, the binding assay reaction mixtures were loaded onto a 6% (w/v) acrylamide gel containing 20 mM magnesium acetate and run at 35°C for 90 min or 60°C for 70 min at 76 V in 1× TAE buffer (15) containing 20 mM magnesium acetate. The gel was then dried and placed in contact with an imaging plate. The bands were analyzed using a BAS2000 image analyzer. When the assay was carried out using 16 bp duplex DNA, concentration of magnesium ion was adjusted to 5 mM in the assay buffer and 1 mM in the running buffer, respectively.