Before the advent of polymerase chain reaction (PCR) technology, many methods for site-directed mutagenesis basically relied on enzymatic extension of a mutagenic oligonucleotide annealed to a single-stranded template and amplification of the ligase-sealed double-stranded heteroduplex in anEscherichia colihost (1 ). Several techniques have been employed to enhance the frequency of clones containing the desired mutation by selecting against the parental strand through biological means or by in vitro manipulations (2 –4 ).
