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Recombinant DNA Technology

rDNA technology  is facilitate to study of genes, they can be isolated and amplified. One method of isolation and amplification of a gene of interest is to clone the gene by inserting it into another DNA molecule that serves as a vehicle or vector that can be replicated in living cells. When these two DNAs of different origin are combined, the result is a recombinant DNA molecule.
The recombinant DNA molecule is placed in a host cell, either prokaryotic or eukaryotic. The host cell then replicates (producing a clone), and the vector with its foreign piece of DNA also replicates. The foreign DNA thus becomes amplified in number, and following its amplification can be purified for further
              Two major categories of enzymes are important tools in the isolation of DNA and the preparation of recombinant DNA: restriction endonucleases and DNA ligases. Restriction endonucleases recognize a specific, rather short, nucleotide sequence on a double-stranded DNA molecule, called a restriction site, and cleave the DNA at this recognition site or elsewhere, depending on the type of enzyme.
              DNA ligase joins two pieces of DNA by forming phosphodiester bonds. First, the DNA fragments to be cloned are generated by using restriction endonucleases, as described in Section. Second, the fragments produced by digestion with restriction enzymes are ligated to other DNA molecules that serve as vectors. Vectors can replicate autonomously (independent of host genome replication) in host cells and facilitate the manipulation of the newly created recombinant DNA molecule. Third, the recombinant DNA molecule is transferred to a host cell. Within this cell, the recombinant DNA molecule replicates, producing dozens of identical copies known as clones. As the host cells replicate, the recombinant DNA is passed on to all progeny cells, creating a population of identical cells, all carrying the cloned sequence. Finally, the cloned DNA segments can be recovered from the host cell, purified, and analyzed in various ways.
 Methods of Inserting Genes into Eucaryotic Cells
• Microinjection into egg cells
• Electroporation
– Pulses of high voltage (250-4000 V/cm)
• Gene Gun
– Developed at Cornell
-Blast of compress air shoots spray of DNAcoated microprojectile.
                     Cloning vectors are carrier DNA molecules. Four important features of all cloning vectors are that they: (i) can independently replicate themselves and the foreign DNA segments they carry; (ii) contain a number of unique restriction endonuclease cleavage sites that are present only once in the vector; (iii) carry a selectable marker (usually in the form of antibiotic resistance genes or genes for enzymes missing in the host cell) to distinguish host cells that carry vectors from host cells that do not contain a vector; and (iv) are relatively easy to recover from the host cell. There are many possible choices of vector depending on the purpose of cloning. The greatest variety of cloning vectors has been developed for use in the bacterial host E. coli. Thus, the first practical skill generally required by a molecular biologist is the ability to grow pure cultures of bacteria.
                  Two commonly used plasmid vectors are shown above These were artificiallyconstructed for the purpose of isolating and moving pieces of foreign DNA into microbialsystems. pBR322 contains the genes for ampicillin resistance and tetracycline resistance.This means that a microorganism containing this plasmid is capable of growth on mediacontaining both of these antibiotics, while microorganisms that do not contain this
plasmid could not grow on the antibiotic containing media. This antibiotic resistance canbe used as a means of selecting and enriching for only those organisms that contain theplasmid. screening of positive (white) colonies can be done by restriction endonuclease digest to confirm the presence and orientation of the insert . When a positive colony containing recombinant plasmid DNA is transferred aseptically to liquid growth medium, the cells will continue to multiply exponentially. Within a day or two, a culture containing trillions of identical cells can be harvested.
                    The final step in molecular cloning is the recovery of the cloned DNA. Plasmid DNA can be purified from crude cell lysates by chromatography using silica gel or anion exchange resins that preferentially bind nucleic acids under appropriate conditions and allow for the removal of proteins and polysaccharides. The purified plasmid DNA can then be eluted and recovered by ethanol precipitation in the presence of monovalent cations. Ethanol precipitation of plasmid DNA from aqueous solutions yields a clear pellet that can be easily dissolved in an appropriate buffered solution.
                    By using PCR and cDNA techniques we can amplify the recombinant DNA in wanting number.
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