Patent ID: 6673610
Filing Date: 2004-01-06
Classification: C12N,C12Q

Abstract:
A method for mutagenesis comprising:annealing one or more primers having a nucleotide sequence containing at least one mutation and a phosphorylated 5â€²-terminus, to a DNA template; elongating the annealed primer or primers by using a DNA polymerase; ligating the phosphorylated 5â€²-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.