A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR
A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCRAndreas Urban, Susanne Neukirchen and Karl-Erich Jaeger*
Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universität, Universitätsstrasse 150, D-44780 Bochum, Germany
Received March 17, 1997;Revised and Accepted April 2, 1997
ABSTRACT
A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.
Mutagenesis by overlap extension has previously been described as a method for both site-directed mutagenesis to create base substitutions, insertions or deletions (1 -3 ) and production of chimeric genes by combining two DNA fragments without a need for restriction sites (4 ,5 ). In separate PCR reactions, two fragments of a target sequence are amplified by using, for each reaction, one universal and one mutagenic primer. The two intermediate products with terminal complementarity form a new template DNA by duplexing in a second reaction. During this so-called overlap extension the fused product is amplified with the help of the two universal primers. To obtain a high mutant yield, it is necessary to fractionate and purify the products of the first PCR reaction by gel electrophoresis (2 ,5 ).
Here we describe an overlap extension strategy which is reduced to only one PCR reaction (Fig. 1 ) and is therefore named one-step overlap extension PCR (OOE-PCR). The target DNA for mutagenesis must be present in both orientations relative to one universal primer. Therefore, it has to be cloned into two vectors which only differ with respect to the orientation of their multiple cloning site with pBluescript II KS and SK or pUC18 and 19 as prominent examples. This cloning strategy has previously been used in a study to optimize the widespread megaprimer PCR method (6 ). The DNA fragments cloned into these plasmids serve as templates in a single PCR reaction in the presence of one universal and two mutagenic primers. Both mutagenic primers B and C are used in a 100-fold dilution as compared to the universal primer A. During the first PCR cycles, the intermediate product AB is amplified exponentially with primers A and B which bind to the template T1. In the same way, the product CA is amplified by binding of primers C and A to template T2. Since neither template exhibits terminal complementarity, wild-type DNA is not amplified. The hybridization of primer A to template T2 and primer C to T1, respectively, should also not lead to an amplification of the wild-type DNA sequence. Due to their high dilution, the mutagenic primers are used up during the early PCR cycles resulting in an asymmetric amplification of the intermediate products with those strands accumulating which form the duplex. The terminal complementarity of the mutagenic primers then leads to formation of the dimer BC thereby further withdrawing primers from the reaction and supporting the asymmetry of the amplification reaction. In this way, a competition between overlapping of the intermediate products and renaturation of these double strands favour the formation of the duplex DNA ABCA. A self-annealing of intermediate products can reduce the final product yield and has been observed for both the overlap extension and the megaprimer method (7 ,8 ). Introduction of an additional step of asymmetric synthesis was proposed to solve this problem (9 ,10 ). The fused mutant product AA contains one universal primer-binding site each at both ends and is therefore amplified as soon as it is formed in the late cycles of the PCR reaction. We tried to further simplify the OOE-PCR by using only one mutagenic and one universal primer which would also make this method less expensive. Although we obtained detectable products, the overall yield of mutant DNA was very low.
