Q.Explain the steps in the process of rDNA technology with suitable diagrams.
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Start your 14-day free trial to unlock the full solution →Step 1 -- Isolation of DNA from the donor organism. Donor cells are sheared and treated with a detergent to release their genetic material, which is purified; it is then cut with a restriction enzyme into fragments, and the fragment carrying the desired gene (now called foreign/passenger DNA) is selected. It can also be sourced directly from a genomic or cDNA library.
Step 2 -- Insertion into a cloning vector. The vector (commonly the plasmid pBR322) is isolated and cut with the same restriction enzyme used on the donor DNA, so the cut ends match. DNA ligase then joins the foreign DNA into the vector, forming recombinant (chimeric) DNA.
Step 3 -- Transfer into a competent host. The recombinant DNA is introduced into a competent host cell (usually a bacterium such as E. coli) by transformation, a process assisted by Ca2+ ions; the host incorporates the rDNA into its own chromosome, becoming a transformed cell.
Step 4 -- Selection of the transformed host cell. Because transformation gives a mixed population of transformed and non-transformed cells, marker genes on the vector (e.g. the ampicillin-resistance gene on pBR322, disrupted when PstI is used) are used to identify and isolate the true recombinants.
Step 5 -- Multiplication of the transformed host cell. Selected transformed cells are grown in fresh culture medium, where they divide repeatedly, replicating the recombinant DNA along with their own genome. …
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