title: [A method for introducing mutations into large vectors].
abstract: OBJECTIVE
  In vitro site-directed mutagenesis is a routine technique in molecular biology labs. However, although there are numbers of related methods available, most of these methods are not suitable for introducing mutations into large vectors.
  
  
  METHODS
  In this report, we describe a method which is highly effective for this purpose. Our method is based on the other site-directed method we recently reported. The basic protocol of our method is as follows: (1) Synthesize a pair of vector primers based on the sequences around the region to be mutated, each containing a suitable type IIs endonuclease restriction site; meanwhile, synthesize a pair of short complementary oligonucleotides which forms a mutagenic fragment after annealing; (2) Synthesize a pair of bridge primers which can specifically bind to a site in the vector sequence, each containing a suitable type IIs endonuclease restriction site; (3) Perform PCR reactions using these Vector primers and Bridge primers; (4) Digest the PCR products with the corresponding type IIs restriction enzyme; (5) Ligate the digested fragment with the mutagenic fragment to make the desired mutant.
  
  
  RESULTS
  Using this protocol, we have introduced mutations into a vector larger than 9 kb. The results shows that the mutation rates are more that 90%.
  
  
  CONCLUSIONS
  Our method provides a useful tool for performing site-directed mutagenesis experiment in large vector.
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute,  Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Fanrong
authors.initials: F
authors.lastname: Meng
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute,  Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute,  Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Haisu
authors.initials: H
authors.lastname: Wan
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviroment, Tianjin Lung Cancer Institute,  Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Zhou
mesh_terms: D017931:DNA Primers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers