PCR-Assisted Mutagenesis for Site-Directed Insertion/Deletion of Large DNA Segments
We present in this chapter a polymerase chain reaction (PCR)-based method to simultaneously introduce and remove large fragments of DNA in a single mutagenesis reaction without the need for restriction sites. We have favored the use of long single-stranded DNA primers synthesized by asymmetric PCR (1,2), whereas others have used double-stranded PCR-amplified fragment directly (3, 4, 5) as primers in in vitro mutagenesis reactions.
KeywordsHelper Phage Initial Polymerase Chain Reaction Restriction Enzyme Cleavage Site Asymmetric Polymerase Chain Reaction Mutagenesis Reaction
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