Method for effecting site-directed mutagenesis

Abstract

A method for performing site-directed mutagenesis characterized in that the method includes the step of carrying out PCR by the use of a double-stranded DNA vector having one or more amber codons, the vector resulting from insertion of a target DNA fragment for site-directed mutagenesis, and at least two kinds of selection primers; and a kit for site-directed mutagenesis for use in the above method, characterized in that the kit includes amber codon reversion primers. According to the present invention, there can be provided a method for performing site-directed mutagenesis and a kit, which is useful for genetic engineering or protein engineering, more simply and rapidly. By using the method and the kit of the present invention, it is possible to efficiently obtain a mutation-introduced gene at the desired position by simply transforming a host with a PCR product obtained by PCR.

1 . A kit for site-directed mutagenesis comprising a termination codon reversion primer, PCR reagents, a thermostable DNA polymerase suitable to carry out LA (Long and Accurate) PCR, and a suppressor-free (Sup 0 ) host, wherein said suppressor-free (Sup 0 ) host is  Escherichia coli.    
     
     
         2 . The kit for site-directed mutagenesis according to  claim 1 , wherein said kit further comprises a double-stranded DNA vector having at least one termination codon and a multicloning site for target DNA insertion.  
     
     
         3 . The kit for site-directed mutagenesis according to  claim 2 , wherein said double-stranded DNA vector is selected from the group consisting of: pKF18k-2, pKF19k-2, pKF19kM and pKB101.  
     
     
         4 . The kit for site-directed mutagenesis according to  claim 2 , wherein said termination codon is located on a drug resistance gene of said vector.  
     
     
         5 . (Canceled).  
     
     
         6 . A kit for site-directed mutagenesis comprising a termination codon reversion primer, PCR reagents, a thermostable DNA polymerase, suitable to carry out LA (Long and Accurate) PCR, a suppressor-free (Sup 0 ) host, wherein said suppressor-free (Sup 0 ) host is  Escherichia coli , and optionally a double-stranded DNA vector having at least one termination codon and a multicloning site for target DNA insertion 
 wherein said kit allows for rapid 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 an 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 PCR 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 the suppressor-free (Sup 0 ) host with the PCR product of step (1), thereby directly obtaining a desired mutant.