A universal nucleoside for use at ambiguous sites in DNA primers

A NON-DISCRIMINATORY base analogue, or universal base, would be an invaluable component of oligonucleotide probes and primers for solving the design problems that arise as a result of the degener-acy of the genetic code, or when only fragmentary peptide sequence data are available. We have designed an alternative to previous universal nucleoside candidates^1-9, a new analogue, l-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole (designated M; Fig. 1), which max-imizes stacking while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex. Oligonucleotides con-taining M at several sites were used as primers for sequencing and the polymerase chain reaction. The sequencing primer d(5′-CGT AAM CAM AAM ACM AT-3′) is as effective as the exact match d(5′-CGT AAT CAG AAA ACA AT-3′). It is also possible to sequence using a primer containing M at several contiguous posi-tions, for example d(5′-CGT AAT MMM MMM MMM AT-3′). Melting curves show that duplexes formed on hybridization of the sequences d(5′-CCT TTT TMT TTT TGG-3′) and d(5′-CCA AAA AXA AAA AGG-3′), where X is A, C, G or T, melted at a lower temperature than the corresponding duplexes containing only d(A.T) and d(C.G) base pairs, but showing little variation among different X bases (T_m range 3°C).