DNA may be manipulated in the test tube and the altered DNA construct may then be inserted into the target organism. The simplest form of such in vitro mutagenesis is
Site-Directed Mutagenesis 367
TABLE 13.02 Techniques used for In Vitro Mutagenesis Chemical mutagenesis of cloned DNA
The gene to be mutagenized is cloned onto a suitable vector, usually a plasmid. DNA carrying the target gene is extracted and purified and treated with a chemical mutagen in vitro. The altered DNA is then transformed back into the original organism and screening is carried out to identify organisms that received a mutant version of the gene. Gene disruption by restriction and ligation (See Ch. 22, Recombinant DNA) A DNA cassette, often carrying a gene for resistance to some antibiotic to allow selection, is inserted into the target gene by using restriction enzymes and DNA ligase. This approach is often used if convenient restriction sites are available. If not, then PCR-based introduction of extra DNA is a good alternative.
In vitro DNA synthesis (See Ch. 24, Genomics)
Single stranded DNA is sometimes generated for sequencing by using M13 vectors. In vitro DNA synthesis may be performed using such ssDNA as template using T7 polymerase and a supply of nucleoside triphosphates. DNA polymerization may be initiated using artificially synthesized primers whose sequence has been altered by a few bases. This will generate a mutagenized product that incorporates these changes. This technique has largely been replaced by PCR based methods.
PCR based techniques (See Ch. 23, PCR)
a) Introduction of Specific Base Changes
Using PCR primers whose sequence has been altered will generate a PCR product that incorporates these changes.
b) Localized random mutagenesis
Manganese ions cause errors in PCR reactions. Hence random mutations may be introduced into the segment of DNA being amplified.
c) Generation of Insertion or Deletion by PCR
Using PCR primers that include sequences homologous to the target location allows replacement of a region of chromosome with a segment of DNA generated by PCR.
Transgenic technology creates genetically modified organisms. It may therefore be regarded as a form of mutagenesis. Extra DNA sequences may be introduced from other organisms, by a variety of techniques.
DNA alterations are often constructed by a variety of genetic engineering techniques.
to treat purified DNA with mutagenic chemicals or radiation. However, a variety of more sophisticated techniques have been used to deliberately construct mutations utilizing genetic engineering technology. These techniques are usually known as directed mutagenesis (or, sometimes, site-directed mutagenesis, when the site of mutation is carefully controlled). Some techniques introduce changes in one or a few bases, whereas others involve more drastic alterations. Some in vitro techniques generate semi-random base changes whereas others are extremely specific. Assorted methods have been used, in particular PCR (see Ch. 23) is now widely used and has replaced many of the older approaches. These applications are discussed together with the appropriate techniques later in this book and are summarized here for reference in Table 13.02.
directed mutagenesis Deliberate alteration of the DNA sequence of a gene by any of a variety of artificial techniques site-directed mutagenesis Deliberate alteration of a specific DNA sequence by any artificial technique
Was this article helpful?