[A method for introducing mutations into large vectors].

BACKGROUND AND

objectiveIn 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.

methodsIn 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.

resultsUsing this protocol, we have introduced mutations into a vector larger than 9 kb. The results shows that the mutation rates are more that 90%.

conclusionsOur method provides a useful tool for performing site-directed mutagenesis experiment in large vector.