A method for introducing mutations into large vectors

BACKGROUND AND 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.