Improved site-directed mutagenesis method using PCR. Nucleic Acids Res

Several methods for site-directed mutagenesis using PCR have been described in the last few years. One of the most rapid, universal and economical methods was described by Landt et al. (1). This procedure requires just one mutagenic primer and two universal primers, which may contain convenient restriction sites for cloning. Essentially, it makes use of two subsequent amplification rounds, the first with the mutagenic oligonucleotide and the antiparallel universal primer and the second one using the purified first fragment as a primer, together with the second universal primer, and subsequent digestion and cloning of the fragment. A possible problem described by these authors is the untemplated addition of one nucleotide at the 3 ' site-specifically altered end of the first amplified fragment by Tag-poly merase, which can give rise to unwanted mutations in the second generated fragment. The authors advise to use lower concentrations o