Mutation in genes often produces alterations in phenotype. To identify a gene that has been mutated and where the phenotype has been changed consequently, it would be necessary to identify a gene on the basis that it carries a mutation. This is the main idea behind gene tagging. A number of molecular tags have now been determined by many genes in most of the plants. These molecular tags include cloned restriction fragment length polymorphism (RFLP) probes, oligonucleotides RFLP probes, variable number of tandem repeats (VNTR), microsatellite, minisatellite, DNA fingerprint loci. These markers must satisfy the given criteria to be used as tags. Gene tagging and marker assisted selection is an essential component of molecular breeding and is based on the saturation mapping of the genome. This has opened up the possibilities of identifying, mapping, tagging and even isolating or transferring quantitative trait loci (QTL). Thus the most powerful application of DNA markers in plant breeding might help in cloning genes. Earlier, cloning such genes was difficult. With the invention of DNA markers and transposon tagging, important genes have now become accessible to molecular cloning. DNA markers provide the essential starting point for physical isolation of genomic regions containing the gene of interest. The efforts that are involved in tagging a gene can be used further as a part of marker assisted selection programme. as economically important genes are tagged, they can even be transferred to unrelated species.
The molecular isolation of transposable elements now permits the cloning of genes in which the element dwells. The major advantage of this system is that the genes whose function is not known can be cloned. In this case, firstly we have to identify a plant that is mutant for a specific trait so that the transposable element can be inserted into it. Next, a genomic library of the plant stock is created. This library is then screened with a clone for the transposable element. A subclone containing sequences from the gene is then developed from the non-transposable elements. The clone is then used to screen the genomic library containing DNA from a normal plant. Thus, any clone selected should contain the normal copy of the gene. Now, the researchers have started using the elements from the corn into other species using transformation techniques. It has been demonstrated that these elements can be induced to move from the required location to another in the new species. If this movement is coupled with the appearance of mutant phenotype, then the gene responsible for the phenotype can be cloned in that particular species. These techniques have now allowed the use of transposon tagging in plant species in which active transposable elements have not been identified.
The mechanism of gene tagging involves four steps. In the first step, a large number of mutations are generated using as mutagen, a piece of mobile DNA that can insert at random in the genome, disrupting and blocking the function of any gene into which it inserts. This results in a population of organisms where the only difference between them is the position of the mutation. The mobile DNA used for the mutagenesis is usually a transposon; that is a piece of DNA which is able to jump to different position in the genome. Ideally, the transposon is one which is not present in the genome of the organism but nevertheless is capable of transposition when introduced into that organism. Usually, the transposition will carry a selectable marker such as an antibiotic resistance marker. This would help in selecting the organisms with transposons in their genome. They are examined to detect the mutant phenotype of interest. Now, DNA is isolated from these organisms and used in making a gene library. Finally, the library is screened to identify the clones which contain the transposons.
With the advancement in molecular biology, the use of molecular markers has given rise to molecular breeding system. Gene tagging and molecular selection is an essential component of molecular breeding and is based on mapping of genomes. This has opened up the possibilities of identifying, mapping, tagging and even isolating and transferring QTL. Thus, significant genes have now become accessible to molecular cloning.
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