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Abstract for Altered Sites® II in vitro Mutagenesis Systems
The Altered Sites® II in vitro Mutagenesis Systems provide a high-efficiency procedure for generation and selection of oligonucleotide-directed mutants. These second generation systems feature several improvements over the original Altered Sites® Systems, including the ability to mutagenize double-stranded template DNA, perform sequential rounds of mutagenesis without subcloning, and express the mutated gene products in vivo or in vitro. Each system uses one of three phagemids.
The systems use antibiotic selection as a means to obtain a high frequency of mutants. For example, the pALTER®-1 Vector contains genes for ampicillin and tetracycline resistance, but the ampicillin resistance gene has been inactivated. The Ampicillin Repair Oligonucleotide restores ampicillin resistance to the mutant strand during the mutagenesis reaction. This oligonucleotide is annealed to the single- or double-stranded DNA template at the same time as the mutagenic oligonucleotide. Subsequent synthesis and ligation of the mutant strand links the two. The appropriate oligonucleotides can be used simultaneously in the mutagenesis reaction to inactivate one resistance gene while repairing the other. In this way, subsequent rounds of mutagenesis and selection can be performed on the same plasmid without subcloning.
The pALTER®-Ex1 Vector, a derivative of the pALTER®-1 Vector, contains appropriate transcription and translation signals for protein expression in vivo or in vitro. The pALTER®-Ex2 Vector is a colE1-compatible vector that can be used in bacteria already transformed with pBR322- or pUC-derived plasmids. The pALTER®-Ex2 Vector has selections for chloramphenicol and tetracycline resistance as well as the appropriate signals for protein expression. With each Altered Sites® II System, the appropriate mutagenic oligonucleotides are provided to both inactivate and repair the antibiotic resistance genes of the supplied vector.