RDT is a series of procedures that is used to join together (recombine) DNA
segments from different organisms and inserting it into a host organism to produce new genetic combinations.

DNA (rDNA) is m
olecules of DNA from two different species that are inserted into a
host organism to produce new genetic combinations that are of value to science,
medicine, agriculture, and industry.

DNA in a living organism was first achieved in 1973 by Herbert Boyer,
of the University of California at San Francisco, and Stanley Cohen,
at Stanford University
, who used  E. coli restriction
enzymes to insert foreign DNA into plasmids.

Using RDT we can get a huge no of copies/clones of a particular gene or
DNA(cloning) by using cloning vectors or we can produce our desired
protein inside a host cell by using expression vectors.



Isolation of the

The 1st step of RDT is the
generation of DNA fragments using restriction enzymes and selection of desired
piece of pure DNA i.e. free from other macromolecules, from chromosome.

Restriction enzymes basically act as molecular scissors(shown in Fig-1) that cut DNA at
specific location.
reactions are called ‘restriction enzyme digestions’. They involve the
incubation of the purified DNA with the selected restriction enzyme, at
conditions optimal for that specific enzyme.

The vector
DNA is also processed using the same restriction
(As each RE cuts in specific locations,so if we treat the vector
with other RE , joining of the dna and vector may not be possible).

Ligation of DNA
molecule and vector:

The 2nd step is insertion of the DNA (to be cloned or
expressed) into a vector(which acts as a vehicle for the selected DNA to get
into the host cell and to be replicated inside the host cell) to form the
recombinant DNA.

The purified DNA and the vector of interest are cut with the
same restriction enzyme,which gives us the cut fragment of DNA and the cut
vector, that is now open.The process of joining these two pieces together using the
enzyme ‘DNA ligase’ is ‘ligation’.

The result­ing DNA molecule is a hybrid of two DNA molecules
– the interest molecule and the vector. In the ter­minology of genetics this
intermixing of dif­ferent DNA strands is called recombination.

Hence, this new hybrid DNA molecule is also called a
recombinant DNA molecule and the technology is referred to as the recom­binant
DNA technology

3. Introduction of the recombinant DNA(rDNA) into the host cell:

In this step, the recombinant DNA is introduced into a
recipient host cell mostly, a bacterial cell. This process is Transformation’. Bacterial
cells do not accept foreign DNA easily. Therefore, they are treated to make
them ‘competent’ to accept
new DNA. The processes used may be thermal shock,
Ca++ ion treatment, electroporation

Isolation of the
clones containing the rDNA:

Now as the host cell divides, with increasing
no of cells the no of rDNA also increases in the medium i.e, we started getting
clones and if these cells contains appropriate elements for expression of the
gene then we get desired product from the cells.

Recombinant DNA Tech is used to produce human insulin:

q  At
first the gene for insulin is isolated from human cell by treating with
appropriate restriction
And plasmids are also cut with the same restriction
enzymes as the DNA,which will allow the DNA to be fixed into the plasmid
DNA ligase


q  The plasmids are then
incubated with a weakened strain of E. coli. Since only some
of the bacteria will take up the plasmid,a gene encoding an enzyme which breaks
down a certain antibiotic is also included in the plasmid, which allows
bacteria with the plasmid to grow on a plate containing the antibiotic while
the other bacteria die. 


q  These bacteria are then
allowed to grow and replicate, which allows the plasmid and the insulin gene to
replicate millions of times. 


q  Then the bacteria are
given a signal to produce the protein, and insulin identical to that of humans
can be produced and purified. 


q  This insulin, which is
abtly named
HUMULIN, can then be used to
treat many people with type I diabetes without the worry of allergic reaction.

What are the
applications of RDT?


DNA is widely used in biotechnology, medicine and research.


The most
common application of recombinant DNA is in basic research, in which the
technology is important to most current work in the biological and biomedical


DNA is used to identify, map and sequence genes, and to determine their


proteins are widely used as reagents in laboratory experiments and to generate
antibody probes for examining protein synthesis within cells and organisms.


additional practical applications of recombinant DNA are found in industry,
food production, human and veterinary medicine, agriculture, and



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