PCR-Assisted Mutagenesis for Site-Directed Insertion/Deletion of Large DNA Segments
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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 –5 ) as primers in in vitro mutagenesis reactions.
