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Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
Genetic engineering/inverse genetics
IX. GENETIC ENGINEERING/INVERSE GENETICS
As described above, genetic mapping involves the ordering of mutations of known phenotypes and provides only vague indication of the actual physical distances involved. Physical analysis of a region of DNA has the great strength of supplying that information and therefore enabling the experimenter to more precisely manipulate the interesting region. More broadly, classical genetics starts with the recognition of phenotypically identifiable variants and then identifies the genotypic mutation that causes that phenotypic difference. Genetic engineering starts with the generation of a particular mutation and then determines its phenotypic effect. Hopefully this text will convince you of the advantages of each methodology and how the intelligent combination of the two is more powerful than either separately.
This section will consider several sorts of analyses leading to the physical ordering of regions of DNA: the relative ordering of restriction fragments, the physical localization of insertion elements in a region, and the application of DNA sequencing to biological questions. As in the sections dealing with classical genetics, the text will not be a lab manual of methods but a discussion of the logic, motivations, and potential results of each method. A final note is that the techniques of classical genetics are largely optimized, but the methodology of physical analysis is improving dramatically and rapidly. It is foreseeable, for example, that DNA sequencing will become so rapid that all other forms of mapping become irrelevant. The development of improved technologies will change not only how we answer questions, but even what questions we choose to address.
Most of the following sections involve the generation of mutations affecting a given gene or gene product without already knowing the resulting phenotype. Obviously, as described below, you need some other information: a purified protein, previously cloned genes, etc. This style of genetics, going after the genotype rather than the phenotype, is rather new and it is still unclear what implications this as well as other new technologies (very rapid automated sequencing; rapid DNA synthesis, etc.) will have on the style of research in biological sciences. To wax philosophical: the experiments we plan and even imagine are limited by our conception of what is reasonably possible to do. Put another way, our methodologies limit our imagination. What needs to be done is to ask "given this technology, what questions are now answerable which I may not have even posed before?" (However, some care has to be given to the problem of asking biologically relevant questions; we cannot be led by the nose by our technology.)
IX A. IDENTIFICATION OF INTERESTING FRAGMENTS (CLONES) OF DNA
IX A1. MUTATION Complementation
The easiest genes to isolate are those for which you can generate a selection. Typically this is done by starting with a mutant organism, lacking the desired gene, and introducing cloned fragments of DNA followed by a selection for the non-mutant phenotype. The source of the cloned gene can either be the wild-type version of the same organism, or one from a completely different organism. The latter is not exactly Complementation, since you do not already know that the "complementing" region actually carries the same gene as that mutated in the chromosome; it could actually carry a suppressor (see XI) of the chromosomal mutation. Further, the introduced region will often be present on a multi-copy plasmid, which again means that it is not exactly Complementation. This scheme can also be used to subclone the relevant region from a large "complementing" clone. For example, if you have a 10-kb fragment on a plasmid that corrects an argA mutation, you could clone smaller fragments into a vector and transform these into the ArgA- strain, selecting Arg+. This approach might fail in the case of small fragments, since it demands that the entire gene and its promoter be functional on the fragment. This approach can be used to "complement" mutations in the same organism or in different ones, but recognize that interspecific complementation can be rather more difficult to interpret.
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