In Vitro Mutagenesis

Another use of cloned DNA is in vitro mutagenesis in which a mutation is produced in a segment of cloned DNA. The DNA is then inserted into a cell or organism, and the effects of the mutation are studied. Mutations are useful to geneticists in enabling them to investigate the components of any biological process. However, traditional mutational analysis relied on the occurrence of random spontaneous mutations—a hit-or-miss method in which it was impossible to predict the precise type or position of the mutations obtained. In vitro mutagenesis, however, allows specific mutations to be tailored for type and for position within the gene. A cloned gene is treated in the test tube (in vitro) to obtain the specific mutation desired, and then this fragment is reintroduced into the living cell, where it replaces the resident gene.

One method of in vitro mutagenesis is oligonucleotide-directed mutagenesis. A specific point in a sequenced gene is pinpointed for mutation. An oligonucleotide, a short stretch of synthetic DNA of the desired sequence, is made chemically. For example, the oligonucleotide might have adenine in one specific location instead of guanine. This oligonucleotide is hybridized to the complementary strand of the cloned gene; it will hybridize despite the one base pair mismatch. Various enzymes are added to allow the oligonucleotide to prime the synthesis of a complete strand within the vector. When the vector is introduced into a bacterial cell and replicates, the mutated strand will act as a template for a complementary strand that will also be mutant, and thus a fully mutant molecule is obtained. This fully mutant cloned molecule is then reintroduced into the donor organism, and the mutant DNA replaces the resident gene.

Another version of in vitro mutagenesis is gene disruption, or gene knockout. Here, the resident functional gene is replaced by a completely nonfunctional copy. The advantage of this technique over random mutagenesis is that specific genes can be knocked out at will, leaving all other genes untouched by the mutagenic procedure.

In gene knockout a functional gene is replaced by an inactivated gene that is created using recombinant DNA technology. When a gene is “knocked out,” the resulting mutant phenotype (observable characteristics) often reveals the gene's biological function.