Scheme 1.
Preparation of a single-stranded DNA template modified with 3-methyladenine (3-meA) within an A-tract. The template strand is annealed to a duplex forming oligomer (DFO) that creates a transient 10 base pair duplex centered at the A4 target sequence. This allows efficient methylation at the 3-position of adenine at 4°C by Me-lex, which selectively methylates double-stranded DNA in the minor groove at A/T rich sequences. The two 5′-A's are heavily and equally methylated at the N3-position. The 3-mA lesion is stabilized within the duplex region and is revealed in the single-stranded template needed for the polymerase studies by warming the reaction temperature to 37°C in the presence of the primer, which results in the melting of the DFO and the annealing of the primer. The reaction is initiated by the sequential addition of polymerase and dNTPs.