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Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
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Mutagenesis in vitro
©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
IV F. in vitro MUTAGENESIS REQUIRING SEQUENCE/SITE INFORMATION
Several of the chemical mutagens listed in Section IIIB can be used for in vitromutagenesis of DNA in a non-specific way. This section deals with a more directed, in vitromutagenesis that requires certain information about the sequence or position of restriction enzyme sites as well as having the DNA region of interest available in purified form. This latter requirement is met by procedures discussed in section VI. The generation of maps of restriction enzyme sites, as well as sequence analysis, will be discussed in Other Types of Mapping. The following schemes allow you to make mutations, not mutants. The latter are generated when the desired mutations are placed on a replicon in vivo, typically replacing the wild-type allele (see end of section).
The possession of purified DNA of a known restriction map can be used to generate site-specific mutations in a variety of ways, but it is first critical to decide what kind of mutations are most useful for you. The following methods allow the generation of either loss-of-function or altered function mutations. The choice of one or another depends on what you already know, and what you want to know, about the gene products.
Linkers: As noted earlier, some restriction enzymes have the property of making staggered nicks in the target DNA. As shown in figure 23, these can be used to add short oligomers, which are commercially available, to the DNA sequence at that site.
If the added bases are not a multiple of three, then a frameshift mutation if created. As noted in the figure, the addition of a particular amino acid or stop signal depends on knowledge of the reading frame of the gene affected. Such linkers can also be used to introduce new restriction enzyme sites.
Cassette mutagenesis: Using the identical scheme outlined above, it is possible to introduce selectable genes at a particular site. Typically, these take the form of drug-resistance genes flanked by multiple restriction sites so that they can ligated into a variety of different target sites.
This system has the advantage over transposon mutagenesis of producing a stable (since it is not a transposable element), absolutely polar (the cassettes have no outward-reading promoters) mutation at physically characterized site. The disadvantage, as with many such in vitro approaches, is that it is much more work, limiting the number of mutants which can be examined. Obviously, one is also limited by the number and position of useful restriction sites.
Deletion formation: By cutting at more than one restriction site in the cloned DNA and religating, it is possible to generate deletions of known end-points as shown in the figure below. While this is most easily accomplished between identical restriction enzyme sites, procedures are described in cloning manuals which allow ligation between virtually any two sites. There are an increasing number of enzymatic activities available that are useful for generation of deletions in vitro.
Site-directed mutagenesis: If the sequence of a region is known, you can generate mutations in an even more precise manner. Oligomers that differ from the wild-type sequence in one or a small number of selected sites are chemically synthesized. These synthesized fragments are then introduced into a vector already carrying the wild-type region by a variety of means (see specialized manuals for details) causing the production of a mutation of pre-determined sequence. This approach is useful when other data (homology to other proteins or a known 3-D structure) identify specific amino acid residues as excellent candidates for serving an important function.
Localized mutagenesis: One of the best Fusions of classical and inverse genetics involves the heavy mutagenesis of a small portion of a cloned region, with the subsequent screening for phenotypic variants. The heavy mutagenesis might be accomplished through the deliberate random mis-synthesis of the region in vitro, with the subsequent incorporation of these into a cloned fragment as above. This approach combines the specificity of inverse genetics with the classical phenotypic identification of mutants. It relies on the idea that sequence analysis might reveal an interesting region of a gene product, but might not identify specific amino acids for site- directed alteration.
Making mutants: In all of these above cases, the constructed vector can be used directly or can be moved into the chromosome. This latter operation is easy in the case of the drug-resistant cassettes, since they provide their own selection. Reintroduction of the other classes can be done either by adding a linked, selectable marker into the cloned region or by introducing the cloned region into the cell, allowing recombination to occur without selection, and screening isolated colonies for the appropriate recombinant. If there is reasonable amounts of homology (greater than 500 base pairs) on either side of the generated mutation, such recombination is reasonably frequent and such a construction is not as difficult as might appear. [See sample problems 8 and 9]
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