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

Abstract

A single-stage polymerase-based procedure is described that allows extensive modifications of DNA. The version described here uses the QuikChange Site-Directed Mutagenesis System kit supplied by Stratagene. The original protocol is replaced by a single-stage method in which linear production of complementary strands is accomplished in separate single primer reactions. This has proved effective in introducing insertions and deletions into large gene/vector combinations without subcloning. © 2005 Humana Press Inc. All rights of any nature whatsoever reserved.