Primer-Directed Mutagenesis of an Intact Plasmid by Using Pwo DNA Polymerase in Long Distance Inverse PCR

摘要

Oligonucleotide-directedmutagene-sis is an important method for modifying specific bases of a DNA sequence (8) Current approaches include combining inverse polymerase chain reaction (PCR) with long PCR to introduce mutations directly into an intact plas-mid template (1,5,10). However, the use of Taq DNA polymerase in these protocols results in a high rate of base mis-incorporation (3,7) Moreover, the inherent terminal transferase-like activity of the enzyme results in 3' overhangs that must be blunt-ended before ligation (2). Screening of the clones is also laborious, if direct selection of the desired mutations is not possible.