Abstract: The invention relates to novel chimeric phosphoramidate oligonucleotides and their use in primer-extension methods such as DNA sequencing and nucleic acid amplification. The subject chimeric phosphoramidate oligonucleotides have both N3'-phosphoramidate linkages and phosphodiester linkages. The invention includes methods of primer extension using the subject chimeric oligonucleotides as primers. Primer extension methods of interest include nucleic acid amplification reactions, e.g. PCR, and polynucleotide sequencing reactions. In the primer extension methods of the invention, a chimeric phosphoramidate oligonucleotide primer is annealed to a polynucleotide template. After annealing, the chimeric oligonucleotide primer is extended by joining a nucleotide to the 3' end of the primer by a DNA polymerase catalyzed reaction. Other embodiments of the invention include methods of primer extension using phosphoramidate linkage containing polynucleotide templates.