A universal nucleoside for use at ambiguous sites in DNA primers

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Abstract
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 candidates1–9, 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 (Tm range 3oC).

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