1. Isolation of a useful or desired DXA segment (gene) from a donor cell.
2. Splicing or joining it to a suitable vector under conditions to ensure that each vector receives only one DXA fragment or gene to create a recombinant DXA.
3. Cloning the recombinant DXA in a suitable host cell to produce multiple copies and screening of transformed bacterial colonies carrying the recombinant plasmid
4. Transfer of cloned DXA to other hosts, or
5. Construct a gene library or use the recombinant DXA for expression in a suitable host.
Target gene (Gene of interest) Break Cell
1. The DXA is isolated in pure form from the source cell and then a particular DXA segment having the gene of interest is isolated. At first the DXA of the source is cut with a particular RE and then the DXA fragments are separated by electrophoresis.
In this procedure, DXA fragments are separated into specific bands on agarose gel as per their molecular weight. Then, the band containing the DXA segment of interest is identified by probes. Once the band of DXA fragment is identified, it is eluted from the gel in buffer.
These probes are suitably designed small pieces of single stranded DXA or RX/\ complementary to a segment of desired gene. In a suitable procedure known as Sourthern Blotting, the separated DXA bands are transferred to a synthetic nitro-cellulose sheet from the gel. The DXAs are denatured and allowed to anneal with fluorescent labeled probes.
These probes, then, indicate the particular band containing the DXA of interest. Sometimes, the gene of interest becomes difficult to be isolated from genomic source. In such cases C-DXA (complementary DXA) are used for cloning. C-DXA is obtained by reverse transcription of mRXA. If both the earlier described procedures become unsuitable for getting the gene of interest, then, some genes can be artificially synthesized and cloned. DXA synthesizers are used for automated artificial DXA synthesis.
2. All plasmids used in gene cloning are genetically engineered and are commercially available. Following isolation of genes, the cloning plasmids are cleaved with the same RE used to isolate the gene of interest.
This generates the same cohesive ends in the genes to be cloned and in the cloning plasmids. Then, the gene of interest (inserts) and plasmids are mixed, so that the overhanging cohesive ends in both undergo complementary base pairing (annealing).
The enzyme DXA ligase form phosphodiesters between the DXA fragment and plasmid. This results in the formation of recombinant / chimeric plasmid.
3. The recombinant plasmids are introduced into competent host bacterial cells for cloning. The uptake of DXA by the host is facilitated by the use of divalent cations like calcium in appropriate concentration.
There is chance that some bacterial cells may not take up any plasmids, some may take up non-recombinant plasmids and some may take up the recombinant plasmids (transformed). The desired bacterial cells are screened out with the help of the markers present in the plasmids.
The restriction sites for insert in the plasmids are so designed that the insert is incorporated inside the marker gene in the plasmid. As a result, the recombinant plasmid loses the property of that particular marker (for example resistance to ampicilin), a process known as insertional inactivation.
The bacterial colony developed from bacterium which does not take up any plasmid shows no resistance to any of the two antibiotics. Hence, its sub-culture will not survive in the media containing either of the two antibiotics. The colony developed from bacterium that takes up recombinant plasmid loses resistance to one of the antibiotics due to insertional inactivation. Its subculture survives in the medium containing one of the
antibiotics and does not jvive in the medium containing the other antibiotic.
On the other hand, the colony developed from the bacterium taking up non recombinant plasmid shows resistance to both the antibiotics and its subculture survives in both the media. After the bacterial colonies are grown on a culture medium each colony is screened for the marker character and the colony of interest is identified. Then that particular colony is perpetuated and the other colonies are not preserved.
Some plant cells are not susceptible to Ti plasmid. These plant cells are treated with enzyme cellulase to covert them to protoplasts. The protoplasts and animal cell are transformed in one of the physical methods described bellow:
(a) Microinjection: This is the method of vectorless direct delivery of recombinant DNA into plant and animal cells particularly mammals. The foreign DXA is delivered into the nucleus with the help of microinjection or micropipette.
(b) Electroporation: This procedure is mostly used for transforming plant protoplasts. The plant protoplasts are placed in suitable medium containing foreign DNA and brief pulses of high voltage electric currents are passed through the medium. These electric pulses create transient openings in the plasma membranes, through which the foreign DNA enter the protoplasts. After the transformation the cell wall is regenerated. E. coli cells those cannot be transformed by cold calcium chloride treatment can also be Firing pin and transformed in this method gunpowder charge
(c) Microprojectile bombardment:
This method is also known as biolistic or gene gun. In this method, DNA coated with microscopic gold and tungsten particles and precipitated with calcium chloride / spermidine / polyethylene glycol are bombarded or shot into target cells. The coated particles are called microprojectiles and they are shot from a particle gun at a high speed 0f300-600m / sec. The coated particles penetrate the cell wall and membrane of the cell. This is mostly used to transform plant cells.