In the classical genetics in which the main theories and laws were propounded by Gregor Mendel, the basic approach for the study directed from gene to phenotype. This classical form of genetics is often termed as forward genetics. A more advanced form of genetics has been introduced that uses the approach of determining a gene's function from its phenotype. The reverse genetics as the name depicts, goes in a backward direction i.e. from phenotype to gene to study the functions of a gene. To put this in simple words, while forward genetics is based on determination of the genetic basis of a particular trait, reverse genetics is based on determination of phenotype on the basis of the genetic information available.
Main techniques of reverse genetics include gene knock out, gene knock in, gene silencing and RNA interference. Gene knock out techniques basically include the deletion of a particular gene from the given sequence. By knocking out a particular gene or in other terms, deleting it, the function that it produces could be supressed since the gene would not code anymore for the protein responsible for the particular function or trait in question. Since the trait produced by the gene won't be expressed anymore, the function of the gene could be determined. The same technique is applied in gene knock in. In gene knock in, a gene is inserted within a sequence. The protein produced by it, expresses itself and the gene in terms of a particular trait or character. Usually an endogenous exon is replaced by a desired gene. Thus by gene knock in and knock out techniques, particular gene can be selected and their functions could be selected by DNA sequencing methods.
Recently, gene knock out mice have been created that seemed exhibit unusual characters. A plant, Physcomitrella patens, has been genetically knocked out by the application of homologous recombination to develop knock out forms of moss and it has been found to be almost as efficient and suitable as when done in yeast. Various gene libraries have been created based on directed deletion in pant systems. In yeast, direct deletions have been performed with almost every non-essential gene present in the yeast genome by the application of gene knock out.
Another technique called, tilling, is a method that combines a standard technique and strategy of mutagenesis with a certain chemical mutagen [for example ethyl methanesulfonate (EMS)]. This is done by the application of a sensitive DNA-screening technique that is used to identify particular point mutations in a target gene.
Gene silencing is another approach in the continuing series. In gene silencing, a particular gene is silenced which causes no formation of its specific protein that is the protein this gene would usually form under the conditions when it is activated and fully functional and expressed. This results into no further expression of the gene and no appearance of its trait that it would usually produce. In this way a particular gene is silenced in order to study its function and expression. So far, detailed gene silencing studies has been carried out and experimentation has been performed on archae and bacteria in which some particular genes were silenced of their respective genomes.
Clustered regularly interspaced short palindromic repeat (CRISPR) are being utilized for the purpose of gene silencing and they are essential components of nucleic-acid-based adaptive immune systems. These systems have been found to be present in bacteria and archaea. The functioning of Clustered regularly interspaced short palindromic repeat is quite similar to RNA interference (RNAi) pathways in eukaryotes since the immune systems based on CRISPR depend on small RNAs. These small RNAs are used for detection that is entirely sequence-specific and silencing of nucleic acids that are not self, i.e. foreign in nature and it includes certain viruses and plasmids. Thus by utilization of gene silencing and directed deletions in context of point mutations, new strategies can be achieved in the field of reverse genetics. By developing an understanding of how small these RNAs are, the information thus gained can be used to find and destroy foreign nucleic acids. Further advancements may facilitate new insights into the underlying mechanisms of RNA-controlled genetic silencing systems to develop new and better strategies.
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