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Reverse genetics

Reverse genetics is an approach to discovering the function of a gene that proceeds oppositely to how such discoveries typically unfold in classical genetics, or in forward genetics.

Classical and reverse genetics are alike in that, by either approach, investigators typically must deduce the function of a normal gene from the effects that follow from damaging or changing it. Otherwise, the two approaches contrast. By the classical approach, geneticists first look for rare individuals with unusual traits or phenotypes, and then they trace these traits to an underlying faulty allele or gene. Locating the gene on its chromosome is the end point of an investigation.

With the readily performed modern techniques of DNA sequencing and as a result of the sequencing of many whole genomes, many genetic sequences are discovered in advance of any other information about them. To learn the influence a sequence has on phenotype, or to discover its biological function, researchers may engineer a change or disruption in it -- by site-directed mutagenesis, for example, by deletion of a gene by gene knockout (as can be done in some organisms, such as yeast and mice) -- and only afterwards look for the effect of such alterations in the whole organism. The discovery of gene silencing using double stranded RNA, also known as RNA interference (RNAi), and the development of gene knockdown using Morpholino oligos have made disrupting gene expression an accessable technique for many more investigators. So phenotype, rather than the starting point, is in reverse genetics the end point.

An alternative used in organisms such as C. elegans is to randomly induce DNA deletions and select for deletions in a gene of interest. Deletions have been created in every non-essential gene in the yeast genome.

Another reverse genetics technique is the application of RNA interference. RNAi creates a specific knockout effect without actually mutating the DNA of interest. In C. elegans, RNAi has been used to systematically interfere with the expression of most genes in the genome.

While RNA interference relies on systems within the cell for efficacy (e.g. the dicer proteins, the RISC complex) a simple alternative for gene knockdown is Morpholino antisense oligos. While RNAi acts by directing cellular systems to degrade target messenger RNA (mRNA), Morpholinos bind and block access to the target mRNA without requiring the activity of cellular proteins and without necessarily accelerating mRNA degradation. Morpholinos are effective is systems ranging in complexity from cell-free translation in a test tube to humans.

Finally, a more difficult genetics technique is the creation of transgenic organisms that overexpress a gene of interest. The resulting phenotype may reflect the normal function of the gene.


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