Premium
A fast and simple method for simultaneous mixed site‐specific mutagenesis of a wide coding sequence
Author(s) -
Follo Carlo,
Isidoro Ciro
Publication year - 2008
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20070045
Subject(s) - mutagenesis , coding region , genetics , biology , plasmid , site directed mutagenesis , computational biology , nucleic acid sequence , inverse polymerase chain reaction , sequence (biology) , mutation , dna , polymerase chain reaction , gene , multiplex polymerase chain reaction , mutant
Background site‐specific mutagenesis at one or multiple sites has recently become an invaluable strategy in functional proteomic studies and genetic engineering. In the present paper we describe a novel PCR‐based procedure for site‐specific mutagenesis that permits, in a single‐step, all three types of nucleotide sequence mutation (deletion, insertion and substitution). The entire procedure is carried out in one tube and takes about 3–4 h. The method utilizes two primers, one of which is phosphorylated at the 5′‐terminus, that are designed to directly anneal back‐to‐back to the target sequence inserted in a plasmid. For the deletion type of mutagenesis (which has virtually no limit to its extent), primers anneal at the ends of the sequence to be deleted. For insertion and substitution types of mutagenesis the primers bear the mutagenic sequences in a tail. The entire circular plasmid, here tested for a maximum length of 7 kbp, is amplified by inverse PCR. The PCR product incorporates the desired mutagenesis and, after ligation, the plasmid is ready for cloning into bacteria. The method has been proved very efficient for deletions of up to 279 nucleotides, for introducing simultaneous deletions, insertions and substitutions, and for performing alanine scanning over a wide coding region. The procedure is suitable for applications in genetic engineering and for the construction of libraries.