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Nucleic Acids Research, 1995, Vol. 23, No. 22 4677-4682
© 1995

Articles

The synthesis of blocked triplet-phosphoramidites and their use in mutagenesis

Akira Ono+, Akira Matsuda, Jia Zhao1 and Daniel V. Santi1,*

Faculty of Pharmaceuticalm Sciences, Hokkaido University Kita-12, Nishi-6, Kita-ku, Sapporo 060, Japan 1Departments of Biochemistry, Biophysics and Pharmaceutical Chemistry, University of California San FRancisco CA 94143–0448, USA

*To whom correspondence should be addressed

Received July 24, 1995. Accepted October 12, 1995.

A general approach for the synthesis of oligonucleotide- triplet phosphoramidites and the synthesis of four such blocks are described. A strategy was devised to minimize the number of dimer precursors needed for synthesis of a complete set of triplet-amidite blocks encoding all 20 amino acids. Whereas synthesis of 20 triplet-amidite blocks consisting of codon sequences requires 16 dimer blocks, just seven dimer blocks are required to synthesize all required antisense sequences. The antisense sequences are then converted to codons in template mediated replication. Using a mixture of four triplet-amidites and conventional automated solid-phase DNA synthesis, short (6mer) and medium length (30mer) oligonucleotide mixtures were synthesized and analyzed. The latter was replicated in vitro and used as a mutagenic cassette to produce four mutants of Asp 221 in the enzyme thymidylate synthase. The method establishes the direction and utility for the production and use of triplet-amidite blocks in DNA synthesis.

+Present address: Department of Chemistry, Faculty of Science, Tokyo, Metropolitan University, 1–1 Minamiosawa, hachioji, Tokyo 192–03, Japan


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