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 mut S) 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.
