Directed-Mutagenesis and Deletion Generated through an Improved Overlapping-Extension PCR Based Procedure

Generating either single or multiple point mutations and producing deletions and insertions into target DNA sequences are critical and widely used experimental procedures in molecular biological studies. This article presents a modified mutagenesis protocol based on an overlapping-extension PCR amplification method. The procedure benefits from the design of mutagenic primers to generate overlapping megaprimers without the need for intermediate purification to remove unused primers. Unused primers are diluted out during the successive amplification-extension reactions. A key success to this modified method is the use of two flanking primers after the overlapping extension reaction. The use of Pfu DNA polymerase increases, compared with Taq DNA polymerase, amplification accuracy. The proposed procedure represents a simple and efficient method that introduces many types of mutations into specific target DNA fragments and creates either hybrid DNA fragments or internal nucleotide deletions.