THE STABILITY OF DUPLEXES INVOLVING AT AND OR G(4ET)C BASE-PAIRS IS NOT DEPENDENT ON THEIR AT/G(4ET)C RATIO CONTENT - IMPLICATION FOR DNA-SEQUENCING BY HYBRIDIZATION/

Sequencing by the recently reported hybridization technique requires t he formation of DNA duplexes with similar stabilities. In this paper w e describe a new strategy to obtain DNA duplexes with a thermal stabil ity independent of their AT/GC ratio content. Melting data were acquir ed on 35 natural and 27 modified duplexes of a given length and of var ying base compositions. Duplexes built with AT and/or G(4Et)C base pai rs exhibit a thermal stability restrained to a lower range of temperat ure than that of the corresponding natural compounds (16 instead of 51 degrees C), The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and /or G(4Et)C base pairs is mainly due to the sequence effect and not to their AT/G(4Et)C ratio content, Thus N-4-ethyl-2'-deoxycytidine (d(4E t)C) hybridizes specifically with natural deoxyguanosine leading to a G(4Et)C base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d(4Et)C can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modifie d DNA targets were successfully amplified by random priming or PCR tec hniques using d(4Et)CTP, dATP, dGTP and dTTP in the presence of DNA po lymerase, This new system might be very useful for DNA sequencing by h ybridization.