Mutagenesis by the overlap extension PCR has become a standard method of creating mutations including substitutions, insertions, and deletions. Nonetheless, the established overlap PCR mutagenesis is limited in many respects. In particular, it has been difficult to make an insertion larger than 30 nt, since all sequence alterations must be embedded within the primer. Here, we describe a rapid and efficient method for creating insertions or deletions of any length at any position in a DNA molecule. This method is generally applicable, and therefore represents a significant improvement to the now widely used overlap extension PCR method.