Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription -- Data Supplement - Supporting Text -- Proceedings of the National Academy of Sciences

Supporting information for Siddiqui-Jain et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.182256799

Supporting Methods
Polymerase Stop Assay.
Briefly, a reaction mixture of template DNA (77-mer with a Pu27 or mutant Pu27 insert) (50 nM), Tris·HCl (50 mM), MgCl (10 mM), DTT (0.5 mM), EDTA (0.1 mM), BSA (60 ng), and 5´-end-labeled 18-mer template (~18 nM) was heated to 90°C for 5 min and allowed to cool to ambient temperature over 30 min. Taq polymerase in storage buffer containing 100 mM KCl (1 µl) was added to the reaction mixture (giving a final KCl concentration of 10 mM), and the reaction was maintained at a constant temperature (45°C for the mutant Pu27 comparison in Fig. 2D; 60°C for the porphyrin comparison in Fig. 3D) for 30 min. (For the porphyrin comparison, after cooling to ambient temperature, the requisite amount of porphyrin solution was added to the reaction mixture and left at ambient temperature for 30 min prior to the addition of polymerase.) After the addition of 10 µl stop buffer [formamide (20 ml), 1 M NaOH (200 µl), 0.5 M EDTA (400 µl), 10 mg bromophenol blue], the reactions were separated on a preparative gel (12%) and visualized on a phosphorimager. Adenine sequencing (indicated by "A" at the top of the gel) was performed using double-stranded DNA Cycle Sequencing System from Life Technologies. The general sequence for 77-mer template strands was TCCAACTATGTATAC-INSERT-TTAGCGACACGCAATTGCTATAGTGAGTCGTATTA, and the 18-mer template had the sequence TAATACGACTCACTATAG.