Megaprimer Mutagenesis
The megaprimer mutagenesis is a method of molecular biology for the targeted alteration of genetic material ( DNA ) in vitro . Megaprimer mutagenesis is a mutagenesis process based on the use of the polymerase chain reaction (PCR) and a synthetic oligonucleotide (primer) containing a mutation . Characteristic of this method is the creation of a so-called megaprimer as an intermediate product.
When mutations are introduced into a DNA using megaprimer mutagenesis, two PCRs are carried out sequentially. In the first PCR, a mutagenic primer and a flanking primer are used to generate a PCR product of 80 to 400 base pairs, the megaprimer. This is used together with a second flanking primer in the second PCR to synthesize a DNA fragment containing the desired mutation. The megaprimer mutagenesis is considered problematic in comparison to other in vitro mutagenesis processes and gives relatively poor yields.
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- ↑ Sambrook J. , Russell DW (2001). Molecular Cloning, 3rd Ed., Cold Spring Harbor, Cold Spring Harbor Laboratory, pp. 13.31-13.35.