MECHANISM OF DEGRADATION OF 2'-DEOXYCYTIDINE BY FORMAMIDE - IMPLICATIONS FOR CHEMICAL DNA-SEQUENCING PROCEDURES

We describe the reaction of formamide with 2'-deoxycytidine to give py rimidine ring opening by nucleophilic addition on the electrophilic C( 6) and C(4) positions. This information is confirmed by the analysis o f the products of formamide attack on 2'-deoxycytidine, 5-methyl-2'-de oxycytidine, and 5-bromo-2'-deoxycytidine, residues when the latter ar e incorporated into oligonucleotides by DNA polymerase-driven polymeri zation and solid-phase phosphoramidite procedure. The increased sensit ivity of 5-bromo-2'-deoxycytidine relative to that of 2'-deoxycytidine is pivotal for the improvement of the one-lane chemical DNA sequencin g procedure based on the base-selective reaction of formamide with DNA . In many DNA sequencing cases it will in fact be possible to incorpor ate this base analogue into the DNA to be sequenced, thus providing a complete discrimination between its UV absorption signal and that of t he thymidine residues. The wide spectrum of different sensitivies to f ormamide displayed by the 2'-deoxycytidine analogues solves, in the DN A single-lane chemical sequencing procedure, the possible source of er rors due to low discrimination between C and T residues. (C) 1997 Else vier Science Ltd.