Abstract
A method for random mutagenesis using primers synthesized from four phosphoramidite solutiosn that have been “spiked” with a small amount of the other three residues is presented. The random oligonucleotides are used in an improved phosphorothioate mutagenesis technique that allows the entire procedure, including transformations, to be completed in 1 day. The phosphorothioate technique gives a high efficiency of mutagenesis from oligonucleotides with random mutations. A large proportion of the transformants analyzed by sequencing are found to contain random changes. The spiked oligonucleotides give truly random mutagenesis within the sequence defined by the primer. This is a very effective method for determining the functionally important amino acids in a protein when little information apart from the DNA sequence is known.
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