Method for mutagenesis

A method for mutagenesis comprising:

• annealing one or more primers having a nucleotide sequence containing at least one mutation and a phosphorylated 5′-terminus, to a DNA template;
• elongating the annealed primer or primers by using a DNA polymerase;
• ligating the phosphorylated 5′-terminus and the elongated terminus of the primer or primers by means of a DNA ligase to synthesize a circular DNA containing said primer or primers;
• denaturing the circular DNA;
• repeating the reactions of annealing, elongating, litgating, and denaturing to amplify the circular DNA to generate DNA products including multiple copies of a single-stranded circular DNA containing the primer or primers;
• selectively digesting the DNA products other than the single-stranded circular DNA to produce megaprimer fragments;
• annealing said megaprimer fragments to said single-stranded circular DNA;
• elongating the annealed megaprimer fragments by using said DNA polymerase to synthesize a double stranded DNA;
• and
• adding an auxiliary primer complementary to a region adjacent to the nucleotide sequence in which mutations are introduced,
• wherein said auxiliary primer is a T7 primer.

A method for mutagenesis comprising:

• annealing one or more primers having a nucleotide sequence containing at least one mutation and a phosphorylated 5′-terminus, to a DNA template;
• elongating the annealed primer or primers by using a DNA polymerase;
• ligating the phosphorylated 5′-terminus and the elongated terminus of the primer or primers by means of a DNA ligase to synthesize a circular DNA containing said primer or primers;
• denaturing the circular DNA;
• repeating the reactions of annealing, elongating, litgating, and denaturing to amplify the circular DNA to generate DNA products including multiple copies of a single-stranded circular DNA containing the primer or primers;
• selectively digesting the DNA products other than the single-stranded circular DNA to produce megaprimer fragments;
• annealing said megaprimer fragments to said single-stranded circular DNA;
• and
• elongating the annealed megaprimer fragments by using said DNA polymerase to synthesize a double stranded DNA,
• wherein a thermostable high-fidelity DNA polymerase and/or a thermostable DNA ligase are used in synthesizing the single-stranded circular DNA and the double-stranded circular DNA.