Abstract for Erase-a-Base® System
The Erase-a-Base® System is designed for the rapid construction of plasmid or M13 subclones containing progressive unidirectional deletions of any inserted DNA. The system is based on the procedure developed by Henikoff, in which exonuclease III (Exo III) is used to specifically digest insert DNA from a 5´ protruding or blunt end restriction site. The adjacent sequencing primer binding site is protected from digestion by a 4-base 3´ overhang restriction site or by an alpha-phosphorothioate-filled end.
Mutagenesis protocols are provided for both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). The mutagenesis reaction is initially transformed into a repair minus strain of E. coli (ES1301 mutS) to avoid selection against the desired mutation. A subsequent strain transfer into JM109 ensures proper segregation of mutant and wildtype plasmids and results in a high proportion of mutants. The Altered Sites® II Mutagenesis Systems allow consistently high mutagenesis frequencies (often >90%) using dsDNA or ssDNA.