Patent ID: 6448048
Filing Date: 2002-09-10
Classification: C12N

Abstract:
A method for performing site-directed mutagenesis which comprises the steps of:(1) producing a PCR product from a single PCR amplification carried out in a reaction tube, wherein in said tube at least the three following components are present: (a) a double-stranded DNA vector having at least one termination codon and having a target DNA fragment for site-directed mutagenesis, (b) a selection primer that binds specifically to said at least one termination codon, wherein said selection primer (b) is a termination codon reversion primer that is designed to revert the termination codon to a non-termination codon, and (c) a selection primer that binds specifically to said target DNA fragment, wherein said selection primer (c) is a mutagenic primer that is designed to introduce a desired mutation into the target DNA fragment, wherein,said PCR amplification reaction comprises at least 20 cycles, wherein in an early cycle, a mixture of at least two PCR products are produced: (i) a copy of the vector template wherein the desired mutation is present in the target DNA fragment of the copy, and (ii) a copy of the vector template wherein the termination codon is reverted to a non- termination codon in the copy, and, in a later cycle of said PCR reaction, said selection primer (b) binds to said PCR product (i) to produce a PCR product (iii) having both the desired mutation and the reverted non-termination codon, and said selection primer (c) binds to said FCR product (ii) to produce a PCR product (iv) having both the desired mutation and the reverted non-termination codon, so that, the PCR products (iii) and (iv) bind to the DNA vector and serve as a long-chain PCR primer for the amplification of a full-length copy of said DNA vector that possesses both the desired mutation and the reverted non-termination codon, within said single PCR amplification; and (2) transforming a suppressor-free (Sup0) host with the PCR product of step (1), thereby directly obtaining a desired mutant.