title: A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning.
abstract: Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.
authors: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, People's Republic of China.
authors: Haisu
authors: H
authors: Wan
authors: Yongwen
authors: Y
authors: Li
authors: Yu
authors: Y
authors: Fan
authors: Fanrong
authors: F
authors: Meng
authors: Chen
authors: C
authors: Chen
authors: Qinghua
authors: Q
authors: Zhou
mesh_terms: D000409:Alanine; D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D055029:Inverted Repeat Sequences / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D009690:Nucleic Acid Conformation; D050814:Octamer Transcription Factor-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific; D000409:Alanine