Abstract for Altered Sites® II Mammalian Mutagenesis System
The Altered Sites® II Mammalian Mutagenesis System provides a
high-efficiency procedure for the generation and selection of oligonucleotide-directed
mutants. The system provides reagents to mutagenize double-stranded (ds) or
single-stranded DNA (ssDNA) templates and to perform sequential rounds of
mutagenesis without subcloning. Expression of sequences in mammalian cells is
also possible without subcloning.
The system uses antibiotic selection to obtain a high frequency of mutants.
The pALTER®-MAX Vector contains the genes for ampicillin and
chloramphenicol resistance, but the ampicillin resistance gene has been
inactivated. In the mutagenesis reaction, the Ampicillin Repair Oligonucleotide
and the specific mutagenesis oligonucleotide anneal to the same strand of the
DNA template. Subsequent synthesis and ligation of the mutant strand links the
two regions, resulting in restoration of ampicillin resistance and introduction
of the desired mutation. The Chloramphenicol Knockout Oligonucleotide can also
be included in the initial reaction to inactivate the chloramphenicol
acetyltransferase gene, thus allowing for a second round of mutagenesis and
antibiotic selection without the need to subclone.
Mutagenesis protocols are provided for both dsDNA and ssDNA. Initially, the
repair minus strain of E. coli (ES1301 mutS) is transformed to
avoid selection against the desired mutation. A subsequent transfer into strain
JM109 ensures proper segregation of mutant and wildtype plasmids and results in
a high proportion of mutant to wildtype clones.
|
