In?vitro?site-specific?mutagenesis

Mutagenesis is a fundamentally important DNA technology which seeks to change the base sequence of DNA and test its effect on gene or DNA function. The mutagenesis can be conducted in vivo (in studies of model organisms, or cultured cells) or in vitro and the mutagenesis can be directed to a specific site in a pre-determined way (site-directed mutagenesis), or can be random. In the case of in vivo mutagenesis, for example, gene targeting offers exquisite site-directed mutagenesis within living cells (Section 21.3.1) while exposure of male mice to high levels of a powerful mutagen such as ethyl nitrosurea (ENU) and subsequent mating of the mice offers a form of random mutagenesis which can be important in generating new mutants (Section 21.4.1).

In vitro mutagenesis can involve essentially random approaches to mutagenesis, which may be valuable in producing libraries of new mutants. In addition, if a gene has been cloned and a functional assay of the product is available, it is also very useful to be able to employ a form of in vitro mutagenesis which results in alteration of a specific amino acid or small component of the gene product in a predetermined way.

6.4.1. Oligonucleotide mismatch mutagenesis is a popular method of introducing a predetermined single nucleotide change into a cloned gene

Many in vitro assays of gene function wish to gain information on the importance of individual amino acids in the encoded polypeptide. This may be relevant when attempting to assess whether a particular missense mutation found in a known disease gene is pathogenic, or just generally in trying to evaluate the contribution of a specific amino acid to the biological function of a protein. A popular general approach involves cloning the gene or cDNA into an M13 or phagemid vector which permits recovery of single-stranded recombinant DNA (Section 4.4.1). A mutagenic oligonucleotide primer is then designed whose sequence is perfectly complementary to the gene sequence in the region to be mutated, but with a single difference: at the intended mutation site it bears a base that is complementary to the desired mutant nucleotide rather than the original. The mutagenic oligonucleotide is then allowed to prime new DNA synthesis to create a complementary full-length sequence containing the desired mutation. The newly formed heteroduplex is used to transform cells, and the desired mutant genes can be identified by screening for the mutation (see Figure 6.19).

Other small-scale mutations can also be introduced in addition to single nucleotide substitutions. For example, it is possible to introduce a three-nucleotide deletion that will result in removal of a single amino acid from the encoded polypeptide, or an insertion that adds a new amino acid. Provided the mutagenic oligonucleotide is long enough, it will be able to bind specifically to the gene template even if there is a considerable central mismatch. Still larger mutations can be introduced by using cassette mutagenesis in which case a specific region of the original sequence of the original gene is deleted and replaced by oligonucleotide cassettes ( Bedwell et al., 1989).

6.4.2. PCR can be used to couple desired sequences or chemical groups to a target sequence and to produce specific pre-determined mutations in DNA sequences

In addition to long-established nonPCR based methods, site-directed mutagenesis by PCR has become increasingly popular and various strategies have been devised to enable base substitutions, deletions and insertions (see below and Newton and Graham, 1997). In addition to producing specific predetermined mutations in a target DNA, a form of mutagenesis known as 5' add-on mutagenesis permits addition of a desired sequence or chemical group in much the same way as can be achieved using ligation of oligonucleotide linkers (see Box 4.2).

5' Add-on mutagenesis

This is a commonly used practice in which a new sequence or chemical group is added to the 5' end of a PCR product by designing primers which have the desired specific sequence for the 3' part of the primer while the 5' part of the primer contains the novel sequence or a sequence with an attached chemical group. The extra 5' sequence does not participate in the first annealing step of the PCR reaction (only the 3' part of the primer is specific for the target sequence), but it subsequently becomes incorporated into the amplified product, thereby generating a recombinant product (Figure 6.20A). Various popular alternatives for the extra 5' sequence include: (i) a suitable restriction site which may facilitate subsequent cell-based DNA cloning; (ii) a functional component, e.g. a promoter sequence for driving expression (see Figure 17.9 for an example); a modified nucleotide containing a reporter group or labeled group, such as a biotinylated nucleotide (see Figure 10.24 for an example) or fluorophore.

Mismatched primer mutagenesis

The primer is designed to be only partially complementary to the target site but in such a way that it will still bind specifically to the target. Inevitably this means that the mutation is introduced close to the extreme end of the PCR product. As described in Section 6.2.3 this approach may be exploited to introduce an artificial diagnostic restriction site that permits screening for a known mutation. Mutations can also be introduced at any point within a chosen sequence using mismatched primers. Two mutagenic reactions are designed in which the two separate PCR products have partially overlapping sequences containing the mutation. The denatured products are combined to generate a larger product with the mutation in a more central location (Higuchi, 1990; Figure 6.20B)

Figure 6.20. PCR mutagenesis. (A) 5' add-on mutagenesis. Primers can be modified at the 5' end to introduce, for example, a labeled group (Figure 10.24), a sequence containing a suitable restriction site (Figure 20.12) or a phage promoter to drive gene expression. (B) Site-specific mutagenesis. The mutagenesis shown can result in an amplified product with a specific pre-determined mutation located in a central segment. PCR reactions A and B are envisaged as amplifying overlapping segments of DNA containing an introduced mutation (by deliberate base mismatching using a mutant primer - 1M or 2M). After the two products are combined, denatured and allowed to reanneal, the DNA polymerase can extend the 3' end of heteroduplexes with recessed 3' ends. Thereafter, a full length product with the introduced mutation in a central segment can be amplified by using the outer primers 1 and 2 only.