A single-stage polymerase-based protocol for the introduction of deletions and insertions without subcloning (original) (raw)

A novel single-stage polymerase-based procedure is introduced that allows for extensive DNA modifications without the need for subcloning. This method replaces traditional protocols with a more efficient linear amplification approach, utilizing the QuikChange Site-Directed Mutagenesis System kit. Comparisons with existing mutagenesis procedures demonstrate advantages in reducing undesirable byproducts, achieving higher specificity, and successfully validating mutagenesis through sequencing. Specific templates and primer designs are outlined for effective transformation and confirmation of mutant products.