PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System

Figure 3

PCR amplification and DNA sequencing of 282-bp DNA templates containing Ds at specific positions. After 25-cycle fusion PCR using the P1 and P4 primers and the 86-bp and 216-bp DNA fragments as templates in the presence or absence of dDsTP and Diol1-dPxTP, the amplified PCR products were analyzed on a 1% agarose gel. Using the purified PCR products, the sequencing reactions were performed in the presence of ddPa′TP (upper sequencing panels) or dPa′TP (lower sequencing panels). The light blue arrow indicates the unnatural base position. The light blue bar indicates the inserted sequence.