4. Oligonucleotide-Directed Mutagenesis using the Polymerase Chain Reaction
New approaches to molecular biology problems are constantly being developed; they are usually simpler, cheaper, and more efficient. Soon after the polymerase chain reaction (PCR) was reported (32), it was realized that it could be adapted to create site-directed mutants (reviewed in (33)). The PCR procedure was developed to amplify a segment of DNA from a minute amount of a DNA sample. The basic method uses two oligonucleotides as primers for DNA synthesis from a DNA template (see PCR). The advantage of using PCR for site-directed mutagenesis stems from its simplicity and speed. The disadvantage of PCR-based methods is the possibility of undesired mutations due to the error-prone nature of certain thermostable DNA polymerases. Today, there are dozens of variations for PCR-based site-directed mutagenesis. Several representative approaches are described here.
4.1. Overlap-extension Method
The overlap-extension method requires four oligonucleotide primers and three separate amplification reactions (34, 35). Two complementary mutagenic primers induce the mutation into the desired sequence of DNA, and two flanking primers amplify the mutant fragment and facilitate cloning of the PCR fragment into a suitable vector. By this approach (Fig. 3), a variety of mutations can be created, such as single base-pair changes, deletions, and insertions. First, two separate PCR reactions are set up in parallel. One reaction has the "sense" mutant primer and an "anti-sense" flanking primer 3′ to the mutation site. The other reaction contains the anti-sense mutant primer and the sense flanking primer 5′ to the mutation site. The two amplified fragments contain mutations at the 5′ or 3′ terminus, respectively. In the second round of amplification, the two fragments from the first round of PCR are purified, then used as templates for amplification using only the flanking primers. After amplification, the mutation is contained within the target DNA segment, which is cloned into appropriate vectors for DNA sequencing and subsequent functional studies.
Figure 3. Site-directed mutagenesis by overlap-extension PCR. The two first rounds of PCR produced two overlapping fragments of the original template, both containing the mutation within the overlap region. These two PCR products are annealed and then subjected to a second round of PCR to generate the entire fragment with the mutation. The flanking primers contain the restriction sites for ligating the fragment back into the original vector.
4.2. Megaprimer Method
The megaprimer method uses only a single mutagenic primer to create mutations in the target template (36-38) (Fig. 4). In the first round of amplification, the wild-type template is amplified using either a sense or anti-sense mutagenic primer and an appropriate flanking primer. The amplified product is then used in a second round of PCR with wild-type template and the other flanking primer to create a fragment of the same length as the original target DNA containing the desired mutation. The key to this method is that the amplified product from the first round of PCR is used as a primer in the second round of PCR. Compared to the four-primer method, this procedure requires only a single mutagenic primer and yields more of the full-length product. This is probably due to the instability of the 10- to 20-basepair overlap between the two mutant templates during the second round of amplification when using the four-primer method. In the megaprimer method, the overlap between the template and mutagenic strands is more extensive.
Figure 4. Site-directed mutagenesis by the megaprimer PCR method. The first round of PCR is used to make a fragment of the template DNA containing the desired mutation. This "megaprimer" is then hybridized to the wild-type template DNA, and a second round of PCR is carried out, to generate the entire molecule with the mutation. The flanking primers contain the restriction sites for cloning the fragment back into the vector.
4.3. Inverse PCR
A third approach, termed inverse PCR, uses only two primers to create the desired mutation (39) (Fig. 5). The key feature of this method is that in making the mutation, the entire vector is amplified. The two primers, one containing the desired mutation, extend on the circular template DNA in opposite directions. Amplification ultimately yields a linear, double-stranded DNA molecule containing the mutation at one end. Following amplification, the ends are ligated, and the resulting circular DNA molecule is transformed into E. coli. There are a number of variations of this method that improve the efficiency of mutagenesis (reviewed in 33).
Figure 5. Site-directed mutagenesis by inverse PCR. The vector is cleaved by a restriction enzyme at a single site, close to the site to be mutated. Two primers from the two ends of the linearized molecule, one containing the desired mutation, are used to amplify the linear molecule and produce molecules with the mutation. After ligating the ends to regenerate a circular molecule, the vector is used to transform E. coli.
The major disadvantage of PCR-based in vitro mutagenesis methods is the lower fidelity of Taq DNA polymerase in DNA polymerization than the phage polymerases used in the traditional methods. This can result in undesired mutations being incorporated into the amplified DNA fragment due to nucleotide misincorporation. If this happens in an early round of amplification, the undesired mutation will be present in a majority of cloned fragments. One simple way to alleviate this problem is to use fewer rounds of DNA amplification and conditions for the highest fidelity of DNA polymerization (40). Alternatively, the use of Pfu or Vent thermal stable polymerases, which are less error-prone than Taq polymerase, will result in fewer undesired mutations. Regardless of which method is used, the DNA sequence of the entire amplified fragment should be determined to ensure that only the desired changes have been made.
5. Evolution of Site-Directed Mutagenesis Applications
Initially, site-directed mutations were constructed one at a time, using a single oligonucleotide. However, multiple mutations can be generated using a degenerate oligonucleotide containing a mixture of related sequences. For example, an oligonucleotide that was degenerate at three adjacent positions was used to generate a series of proteins containing different amino acids at a selected position (11). Doped oligonucleotides are synthesized by spiking each nucleotide monomer with a small amount of the other three monomers. The resulting oligonucleotide will direct a variety of mutations over a defined window of the wild-type sequence (41, 42). The use of degenerate oligonucleotides is particularly powerful when used in conjunction with a genetic screen or selection (43, 44). A particularly powerful method for protein evolution, termed DNA shuffling., uses the polymerase chain reaction to generate proteins with novel or improved properties (45, 46).
In the 20 years since its original report, site-directed mutagenesis has evolved into a standard tool with which to evaluate gene function. It can be accomplished by a variety of methods, many of which are now commercially available in kit form. Given the ease and speed by which site-directed mutagenesis experiments can be performed, the challenge has shifted from simply being able to construct a mutant to designing good experiments.