Circular site-directed mutagenesis

Abstract

The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double-stranded circular DNA intermediates is transformed in suitable competent host cells and closed circular double-stranded DNA corresponding to the parental template molecules, but containing the desired mutation or mutations of interest, may be conveniently recovered from the transformed cells. The invention also provide kits for site-directed mutagenesis in accordance with methods of the present invention.

1 - 17 . (canceled)  
   
   
       18 . A method of introducing a specific mutation into a DNA molecule for mutagenesis, comprising: 
 annealing a mutagenic primer to a single-stranded DNA molecule for mutagenesis;    synthesizing by means of an amplification reaction a mutagenized DNA strand comprising the mutagenic primer, wherein the amplification reaction comprises use of a thermostable polymerase; and    digesting the DNA molecule for mutagenesis, wherein a selection enzyme mediates the digestion.    
   
   
       19 . The method of  claim 18 , wherein the thermostable polymerase is pfu DNA polymerase.  
   
   
       20 . The method of  claim 18 , wherein the DNA molecule for mutagenesis is methylated.  
   
   
       21 . The method of  claim 18 , wherein the digestion occurs in vivo.  
   
   
       22 . A method of introducing a specific mutation into a DNA molecule for mutagenesis, comprising: 
 annealing a mutagenic primer to a single-stranded DNA molecule for mutagenesis;    synthesizing by means of an amplification reaction a mutagenized DNA strand comprising the mutagenic primer, wherein the amplification reaction comprises ten or more amplification cycles; and    digesting the DNA molecule for mutagenesis, wherein a selection enzyme mediates the digestion.    
   
   
       23 . The method of  claim 22 , wherein the amplification reaction comprises use of a thermostable polymerase.  
   
   
       24 . The method of  claim 23 , wherein the thermostable polymerase is pfu DNA polymerase.  
   
   
       25 . The method of  claim 22 , wherein the DNA molecule for mutagenesis is methylated.  
   
   
       26 . The method of  claim 22 , wherein the digestion occurs in vivo.