3-NITROPYRROLE AND 5-NITROINDOLE AS UNIVERSAL BASES IN PRIMERS FOR DNA-SEQUENCING AND PCR

3-Nitropyrrole and 5-nitroindole have been assessed as universal bases in primers for dideoxy DNA sequencing and in the polymerase chain rea ction (PCR). In contrast to a previous report, we have found that the introduction of more than one 3-nitropyrrole residue at dispersed posi tions into primers significantly reduced their efficiency in PCR and s equencing reactions. Primers containing 5-nitroindole at multiple disp ersed positions were similarly affected; for both bases only a small n umber of substitutions were tolerated, In PCR experiments neither base , when incorporated into primers in codon third positions, was as effe ctive as hypoxanthine, which was incorporated in six codon third posit ions in a 20mer oligomer. However, primers containing up to four conse cutive 5-nitroindole substitutions performed well in both PCR and sequ encing reactions, Consecutive 9-nitropyrrole substitutions were tolera ted, but less well in comparable reactions.