Figure 1. Site directed mutagenesis using PCR. A DNA template containing the sequence to be mutated is amplified using PCR. One PCR primer is located at a distance from the site to be mutated, such as in the vector, while the second primer overlaps the target site. By choosing primers located such that the amplified products can be digested with restriction endonucleases and re-cloned back into the starting template, a variety of mutations can be introduced into the template. See step 3 for details.

Figure 2. Introducing small mutations into a template by recombinant PCR. A DNA template containing the sequence to be mutated is amplified using PCR as in Figure 1, except that in this case two reactions are performed to amplify sequences flanking both sides of the target sequence. This approach enables greater flexibility than the approach illustrated in Figure 1, since there is a reduced requirement for a unique restriction endonuclease recognition sequence to be located adjacent to the target site. The mismatch shown in primers C and D can be a single (or 2–3 base changes, or small deletions or insertions (up to a few bases). Larger deletions can be introduced into a template as illustrated in Figure 3. See step 3 for details.

Figure 3. Hybrid PCR primers displaying reverse complementarity can be used to introduce large or small DNA deletions into a template, or to generate recombinant DNA molecules (see also Figures 4 and 5). In this example, primers A and B are reverse complements of one another (bottom of figure). However, each primer contains at its 3¢ end a unique annealing sequence that delineates the region to be amplified and recombined. Since only half of each primer is able to anneal with the target template, conditions of relaxed stringency must be used in the initial round of PCR. However, in the second, or recombinant round, the primers each contain a doubled length of complementarity with the target sequence to be amplified enabling the use of higher annealing temperatures during the PCR. See step 4 for details.

Figure 4. Introducing large deletions into a template by recombinant PCR. Top: A DNA template containing the sequence to be altered is amplified using PCR as in Figure 2, except that in this example the primers do not overlap and could be located at any distance apart, or even on separate molecules. Primers A and B are the same as shown in Figure 3. If the primers A and B are located in the same gene a deletion can be introduced into the gene. Alternatively, the two PCRs shown (A:C and B:D) can be performed on separate templates to generate hybrid genes containing sequences not normally adjacent to one another. Bottom: After amplifying two targets using primers sets A:C and B:D the two products are combined and amplified using primers C and D to generate the recombinant molecule. See step 4 for details.

Figure 5. Introducing insertions into DNA molecules using recombinant PCR. The principle behind this method is similar to the scheme shown in Figure 4, except that three, rather than two, reactions are performed. Using appropriate primers with reverse complementarity to one another, as indicated in the figure, one can introduce any new sequence in between any two other sequences simply by performing a series of PCRs. See step 5 for details.