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Modern Genetic Analysis Chapter 11. Applications of Recombinant DNA Technology
In Vitro Mutagenesis

One of the most established techniques is sitedirected mutagenesis. By using this method, we can create mutations at any specific site in a gene whose wild-type sequence is already known. This known sequence is used to chemically synthesize short DNA segments called oligonucleotides. Through single-strand hybridization, these oligonucleotides can be directed to any chosen site in the gene. In one approach, the gene of interest is inserted into a single-stranded phage vector, such as the phage M13. A synthetic oligonucleotide containing the desired mutation is designed. This oligonucleotide is allowed to hybridize to the mutant site by complementary base pairing. The oligonucleotide serves as a primer for the in vitro synthesis of the complementary strand of the M13 vector (Figure 11-1a). Any desired specific base change can be programmed into the sequence of the synthetic primer. Although there will be a mispaired base when the synthetic oligonucleotide hybridizes with the complementary sequence on the M13 vector, a few mismatched bases can be tolerated when hybridization takes place at a low temperature and a high salt concentration. After DNA synthesis has been mediated by DNA polymerase in vitro, the M13 DNA is allowed to replicate in E. coli, in which case many of the resulting phages will be the desired mutant. The synthetic oligonucleotide can be used as a labeled probe to distinguish wild-type from mutant phages. Although, at low temperature, the mismatched base will not prevent the primer from hybridizing with both types of phage, at high temperature, the primer will hybridize only with the mutant phage. Oligonucleotides with deletions or insertions will direct comparable mutations in the resident gene. The site-directed method can also be used on genes cloned in double-stranded vectors if the DNA is first denatured.

A knowledge of restriction sites is also useful in modifying a cloned gene. For example, a small deletion can be made by removing the fragment liberated by cutting at two restriction sites (Figure 11-1b). With the use of a similar double cut, a fragment, or "cassette," can be inserted at a single restriction cut to create a duplication or other modification (Figure 11-1c). Another approach is to enzymatically erode a cut end created by a restriction enzyme, to create deletions of various lengths (Figure 11-1d).

The polymerase chain reaction (PCR) also can be used (Figure 11-1e). A primer containing a mutation is used in a first round of PCR. The product of the first round is used as a primer for a second round of PCR, whose product is the mutant gene.

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Site-directed mutagenesis allows various types of directed changes to be made at specific sites in a gene.

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