FIGURE 5.

RT-PCR catalyzable by single enzyme Taq D732N but not wild-type Taq. No reverse transcriptase was included, nor was any manganese ion. Two MS2 phage RNA targets, P and K were amplified from 5 pg phage RNA with 0.1 or 0.05 μl D732N mutant or twice as much volume of wild-type Taq (NEB) in 35 μl reactions for 35 PCR cycles. The amplified products were run in a 2.5% agarose gel stained with ethidium bromide, along with a 100 bp DNA ladder (white/black reversed for clarity as printed). As negative control, no RNA was included for the reactions analyzed on the bottom half of the gel. The size of the clearly and successfully amplified bands are 405 and 544 bp (primer-primer gap sizes 359 and 500).