Primer Extension Assay for G-Quadruplex

Primer (LTR G4 Taq primer, Table S1) was 5’-end labelled with [γ-₃₂P]ATP using T4 polynucleotide kinase at 37°C for 30 min. The labelled primer (72 nM), annealed to the template (36 nM) in lithium cacodylate buffer (10 mM, pH 7.4), was extended with AmpliTaq Gold DNA polymerase (2U/reaction, Applied Biosystem, California) at 47°C for 30 min. Where specified, samples were incubated with G-quadruplex-ligands and 100 mM KCl for 20 min at room temperature and primer extension performed as described. Reactions were stopped by EtOH precipitation, extension products were separated on 12% denaturing gel, visualized by phosphorimaging (Typhoon FLA9000, GE Healthcare).