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 degeneracy of the genetic code, or when only fragmentary peptide sequence data are available. We have designed an alternative to previous universal nucleoside candidates, a new analogue, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (designated M; Fig. 1), which maximizes stacking while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex. Oligonucleotides containing 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 positions, 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 3 degrees C).
Nichols
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Andrews
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Zhang
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Bergstrom
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D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D003853:Deoxyribonucleosides; D004331:Drosophila melanogaster; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009705:Nucleosides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013696:Temperature
D017931:DNA Primers; D003853:Deoxyribonucleosides; D009705:Nucleosides; C087515:3-nitropyrrole 2'-deoxyribonucleoside