The present invention relates to an entirely new method for mutagenesis, which can be applied for introducing certain mutations at certain sites of a nucleotide sequence, or for introducing random mutations at certain sites of a nucleotide sequence.
The method for site-directed mutagenesis is a powerful tool for producing mutants to assess the importance of specific amino acid residues in a protein""s structure and function. As such method for site-directed mutagenesis, a variety of protocols have been established, including those using the polymerase chain reaction (PCR).
Among the PCR-based protocols, the Quik Change(trademark) Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA) (referred to as xe2x80x9cQuik Change methodxe2x80x9d, hereinafter) is widely used. According to the protocol of said Quik Change method, a DNA containing mutations can be obtained through the following three steps of:
1) carrying out the PCR process by use of a pair of complementary primers designed to carry a mutation at a desired site;
2) digesting only the template DNAs (that carry no mutations) in the reaction solution; and
3) performing transformation by use of the DNA fragments which are not digested in the above step of digestion.
In particular, said Quik Change method utilizes, in the step of carrying out the PCR process, a supercoiled, double-stranded (ds) DNA plasmid as a template DNA, and a high-fidelity Pfu Turbo(trademark) DNA polymerase as an enzyme. During the step of the PCR process, the pair of complementary primers is annealed to the dsDNA, followed by an elongation reaction so as to amplify DNA fragments that carry mutations and are nicked between the elongated terminus and the 5xe2x80x2-terminus of the primers.
Subsequently, in the Quik Change method, DpnI endonuclease is caused to act in the reaction solution to specifically cut fully- or hemi-methylated 5xe2x80x2-GATC-3xe2x80x2 sequences. The in vitro synthesized and nicked plasmid DNA including the desired mutation is then transformed into E coli As a result, the plasmid DNA that is nicked will be repaired in E coli and become circular.
Thus, according to the Quik Change method, it is possible to synthesize a DNA including a desired mutation at a desired site. However, the Quik Change method suffers from the following disadvantages:
1) multiple mutations can not be introduced at different relatively separated positions in a nucleotide sequence at the same time, i.e., in this method, it is possible to introduce multiple mutations only in a pair of complementary primers, whereas it is impossible to introduce multiple mutations at remote positions;
2) when introducing a mutation, a pair of synthetic complementary oligonucleotides primers is required, that is, this method requires two primers for introducing a mutation at one site; and
3) since it requires a pair of synthetic complementary oligonucleotides primers, it does not allow random mutagenesis at desired sites using degenerative primers.
The Quik Change method, due to the above problem 1), requires that the aforementioned 3 steps be repeated the number of times corresponding to the number of mutagenesis, when introducing mutations at multiple sites. Consequently, the Quik Change method takes a long time to introduce mutations at multiple sites, leading to an inconvenience of poor efficiency. Additionally, the Quik Change method, owing to the above problem 2), involves high cost in synthesizing or purchasing the pair of synthetic oligonucleotides primers. Moreover, the Quik Change method, due to the above problem 3), can not be used in experimental systems where certain amino acid residues in the analyte protein are mutated into a variety of other amino acids, thus limiting the experimental systems to which it is applicable.
Thus, in view of the problems of prior art mentioned above, an object of the present invention is to provide an entirely new method for mutagenesis, which method being simple, speedy, economical, and widely-applicable.
The method for mutagenesis according to the present invention, having achieved said object, comprises the following steps of:
a DNA synthesis in which one or more primers which have a nucleotide sequence containing at least one mutation and a phosphorylated 5xe2x80x2-terminus are annealed to a template DNA and then subjected to an elongation reaction using a thermostable high-fidelity DNA polymerase, after which the phosphorylated 5xe2x80x2-terminus and the elongated terminus are ligated by means of a thermostable DNA ligase to synthesize a circular DNA containing said primers;
a digestion in which said step of DNA synthesis is repeated several times to amplify the DNA containing said primers, after which at least the DNAs other than the amplified circular DNA are then digested into several fragments; and
a double-stranded DNA synthesis in which, with the several fragments obtained in the above step of digestion as megaprimers, said megaprimers are annealed to said circular DNA synthesized in the above step of DNA synthesis, followed by an elongation reaction performed using said thermostable high-fidelity DNA polymerase.