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Reverse
Genetics
and
Chromosome
Jumping
OR
Hopping
Libraries -
In most cases, we identify the genes by their protein products, which help in the isolation and cloning of a specific gene. However, for many human disorders which are each controlled by a single gene ( e.g. huntington disease, cystic fibrosis, duchenne muscular dystrophy, etc.), the gene product is not known despite intensive investigations. Such genes are cloned by a process often called 'reverse genetics', in which the gene is identified primarily by its map position.
Once the gene is cloned, it can be utilized for getting information about the encoded protein, the gene mutation and the metabolic defect that may be associated with the disease. Since the information about the genetics is coming in the reverse order, this is popularly described as 'reverse genetics'.
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This reverse genetics approach involves the following two steps:
(i) locate the gene to a particular chromosome through RFLP linkage analysis;
(ii) map the position of the gene with respect to molecular marker, both through recombinational analysis (in terms of recombination value in cM = centiMorgan units) and by physical mapping (in terms of kilobase pairs) with pulsed field gel electrophoresis (PFGE) and preparation of restriction map.
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Even if the distance is 1cM, which is the limit of resolution, this will be equivalent to 1,00Q kb. Therefore, if the segment carrying the gene is to be cloned using the linked molecular marker, such a segment will be too large to be cloned. In order to bring' the molecular marker close to the gene of interest, 'chromosome jumping' approach has been utilized.
The technique of 'chromosome jumping' is based on the following steps shown:
(i) depending upon the distance between the gene and the marker, decide about the distance of 'jumps' or 'hopsize' (e.g. 100 kb or 200 kb);
(ii) genomic DNA molecules in the range of selected size (say 80 kb-130 kb in case of 100 kb ‘hopsize' or 160-240 kb for 'hopsize' of 200 kb) are selected through pulsed-field gel electrophoresis;
(iii) for circularization of DNA segments, ligation between two ends of each long linear DNA molecule was allowed using T4 ligase in the presence of supF-);
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(iv) DNA circles obtained in (Hi) above are digested with EcoRI;
(v) the vector λCh3A delta lac, an amber mutated phage vector (supF- is also cut with EcoRI and used for cloning small DNA fragments representing the junctions of the circularized genomic DNA molecules and carrying supF+;
(vi) the cloned DNA fragments obtained in
(v) above represent the jumping library, which can be plated on a bacterial host and screened through the technique of plaque hybridization described earlier.
The above technique of chromosome jumping will help narrowing the gap between the gene and available molecular markers. After several cycles of chromosome jumping followed by cloning the regions that are close to the gene, it will be possible to approach very close to the desired and clone it. It is thus obvious that in the field of 'reverse genetics' using technique of chromosome jumping, it will be possible to isolate, clone and characterize genes, whose gene products are unknown.
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The gene for cystic fibrosis has recently been isolated using this, technique. It could also be shown that 'cystic fibrosis' disease is caused by a single base substitution in this gene. This technique of 'reverse genetics' will become a very active area of research particularly in human genetics in the coming years.
The term 'reverse genetics' has however been redefined recently. The classical genetics, sometimes described as 'forward genetics' involves a study which starts from the phenotype, and follows through the identification of the gene and finally concludes with the isolation and sequencing of the gene.
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In view of this, if 'reverse genetics' has to use steps in the reverse order for a genetic study, then it should start with DNA molecules or clones with unknown effect and should conclude with the determination of the phenotype which it controls. This can be achieved by preparing random genomic DNA clones, which can be subsequently utilized for a genetic study in the reverse order. This will then be described as 'reverse 'genetics'.
This definition of 'reverse genetics' differs from the earlier conventional meaning of this term, where 'reverse genetics' was used to describe cases of genes with ,unknown products, which are isolated ( with the help of phenotype and its linkage with molecular markers) and then studied in some detail
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