1) Gene isolation by complementation
2) Positional cloning
3) Gene isolation by sequence homology
4) Gene isolation by functional genomics approach
The technique requires the genomic DNA/ cDNA library of functional alleles and host strain with deficit in trait/gene of interest. The success of technique depends on coverage of genome in the library. Therefore, library construction should be taken at most care and constructed in 2 different vectors to avoid the systemic exclusion. The each recombinant DNA vector from the library is transferred to host strain which does not carry functional allele of the gene of interest or may be recessive allele/mutant version of gene of interest. Screen/select the transformants by providing the conditions such as the gene of interest (GOI) should be present for transformants survival. Those does not able to complement the mutant host strain will die and those able to complement the mutant host strain will survive. For example, theisolation of histidine gene from yeast, the genomic library transferred to mutant host and a cell/s that survives on minimal medium lacking histidine (His-) that presumed to contain histidine gene of yeast. This particular clone can be sequenced and the complete gene can be obtained. The cloning by complementation does have some pit falls such as desired sequence may contain a region or encode a product that is toxic to the library for host organism and multiple suppression. The presence of multiple copies of gene (high copy number of plasmid) cause abundant amount of the product of one gene, it may compensate for loss of another different gene product. It may overcome the mutant/loss of gene function and cells may survive. It leads to wrong identification. Therefore to overcome this problem the copy number of vector need to be kept in mind.
The positional cloning is the process of identification of physical position/proximity site of a cloned DNA fragment in the genome of an organism to isolate the gene of interest. The technique predominantly use chromosome walking process, where a cloned DNA fragment is a starting point from which the walking proceed step wise (clone by clone) fashion towards the gene of interest. During the walking a fragment of clone will be used as a probe to screen next overlapping fragment. The accuracy of determination of consequent fragment increased by use of markers like RFLP and STS and it will avoid wrong overlapping fragment identification. Therefore, physical marker is essentially required to this technique. To arrive at the site of gene of interest is calculated by measuring the distant / cross over frequency between the probe and gene of interest (GOI) trait. As cross over frequency reduces, it indicates the walking is towards the gene.
Homology search based technique is simplest form of gene isolation. From the evolutionary point the genes are conserved between species or within the species hence the orthologous genes are used to isolate the genes from the desired organism. The primer or probe is designed from orthologous gene to amplify/ hybridize the GOI in the genome / genomic library respectively. Apart, if protein sequence is available instead of DNA than degenerative primers are designed to amplify the GOI.
It involves the disrupting the GOI by a DNA fragment (may be T-DNA/TE) it results in loss of function. Screen for the desired mutant phenotype exhibiting individual and trace back to where the T-DNA / TE is inserted (the process is called gene tagging) and based on this the flanking sequence of TE can be identified and complete gene will be isolated.
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