A general strategy for random insertion and substitution mutagenesis: Substoichiometric coupling of trinucleotide phosphoramidites

John Sondek, David Shortle

Research output: Contribution to journalArticlepeer-review

Abstract

Results from a number of recent studies suggest that amino acid insertion mutations may provide an important alternative to substitution mutations for modifying protein structures and functional activities. To facilitate the use of single-amino acid insertions, we have developed a general strategy for inducing random, in-phase codon insertions across a defined segment of a cloned gene. In brief, a mixture of blocked and protected trinucleotide phosphoramidites is coupled at substoichiometric levels after every third monomer coupling on a conventional solid-state synthesizer. From the heterogeneous mixture of oligonucleotide sequences thus generated, those oligonucleotides that have acquired a single additional codon are purified by urea/PAGE. By using equimolar amounts of GCT and GGT trinucleotides in the oligonucleotide synthesis plus standard oligonucleotide-directed mutagenesis techniques, we have induced as many as 13 different single alanine and glycine insertion mutations into the gene for staphylococcal nuclease in one experiment. On replacement of the 5′-dimethoxytrityl blocking group on the trinucleotide phosphoramidite with an acid-stable blocking group, such as levulinate or fluoren-9-ylmethoxycarbonyl (Fmoc), this same strategy of substoichiometric couplings at codon boundaries should permit the synthesis of complex pools of oligonucleotides for the introduction, with constant efficiency, of every type of amino acid substitution at each codon across a gene segment.

Original languageEnglish (US)
Pages (from-to)3581-3585
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number8
DOIs
StatePublished - 1992

Keywords

  • Mutant proteins
  • Oligonucleotide-directed mutagenesis
  • Site-directed mutagenesis
  • Staphylococcal nuclease

ASJC Scopus subject areas

  • General

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