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

Abstract

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G(4Et)C base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51°C). The 16°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 N4-ethyl-2'-deoxycytidine (d(4Et)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. Modified DNA targets were successfully amplified by random priming or PCR techniques using d(4Et)CTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.