AMBIGUOUS BASE-PAIRING OF THE PURINE ANALOG EOXY-BETA-D-RIBOFURANOSYL)-IMIDAZOLE-4-CARBOXAMIDE DURING PCR

In principle the hydrogen bonding capacities of eoxy-beta-D-ribofurano syl)-imidazole-4-carboxamide (dY), and its n-propyl derivative (dYPr), allow them to pair to all four deoxynucleosides. Their triphosphate d erivatives (dYTP and dYPrTP) are preferentially incorporated as dATP a nalogues in a PCR reaction. However, once incorporated into a DNA temp late their ambiguous hydrogen bonding potential gave rise to misincorp oration at frequencies of similar to 3 x 10(-2) per base per amplifica tion. Most of the substitutions were transitions resulting from rotati on about the carboxamide bond when part of the template. Between 11-15 % of transversions were noted implying rotation of purine or imidazole moieties about the glycosidic bond. As part of a DNA template, dYPr b ehaved in the same way as dY, despite its propyl moiety. These deoxyim idazole derivatives are among the most radical departures from the can onical bases used so far as substrates in PCR and could be used to gen erate mutant gene libraries.