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Modern Genetic Analysis Chapter 11. Applications of Recombinant DNA Technology
Reverse Genetics

"Regular" genetics begins, as we have seen, with a mutant phenotype, proceeds to show the existence of the relevant gene by progeny ratio analysis, and finally clones and sequences the gene to determine its DNA and protein sequence. However, reverse genetics, a new approach made possible by recombinant DNA technology, works in the opposite direction. Reverse genetics starts from a protein or DNA for which there is no genetic information and then works backward to make a mutant gene, ending up with a mutant phenotype.

Often the starting point of reverse genetics is a protein or a gene "in search of a function," or, looked at another way, a protein or a gene "in search of a phenotype." For example, a cloned wild-type gene detected by sequencing as an open reading frame (ORF) of unknown function can be subjected to some form of in vitro mutagenesis and reinserted back into the organism to determine the phenotypic outcome. In regard to a protein of unknown function, the amino acid sequence can be translated backward into a DNA sequence, which can then by synthesized in vitro. This sequence is then used as a probe to find the relevant gene, which is cloned and itself subjected to in vitro mutagenesis to determine the phenotypic effect. This approach will become even more significant as data accumulate in whole genomesequencing projects. Sequencing reveals numerous unknown ORFs, many of which have no resemblance to any known gene, and the function of these genes can be determined by reverse genetics. This type of analysis is underway in Saccharomyces cerevisiae;the complete sequencing of the genome of this organism revealed many unassigned ORFs, which are now being systematically mutated into null alleles to detect a phenotype that might provide some clue about function.

Important tools for reverse genetics are in vitro mutagenesis and gene disruption,also known as gene knockout. One approach is to insert a selectable marker into the middle of a cloned gene and then to use this construct to transform a wild-type recipient. Selecting for the marker yields some transformants in which the disrupted (mutated) gene has replaced the in situ wild-type allele (Figure 11-4). Gene knockouts are also considered later in the chapter.

MESSAGE
Reverse genetics discovers the normal role of cryptic DNA or protein sequences by mutating the sequence in vitro and looking for changes at the phenotypic level.

© 1999 by W. H. Freeman and Company. All rights reserved.