Download CHAPTER 6: RECOMBINANT DNA TECHNOLOGY

CHAPTER 6: RECOMBINANT
DNA TECHNOLOGY
YEAR III PHARM.D
DR. V. CHITRA
INTRODUCTION
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DNA : DNA is deoxyribose nucleic acid . It is made up of
a base consisting of sugar, phosphate and one nitrogen
base.The nitrogen bases are adenine (A) guanine (G)
cytosine(C) and thymine(T). The nitrogen bases are
grouped as
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PURINES : Adenine and guanine.
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PYRAMIDINES : Cytosine, uracil and thymine.
The sequence of bases are in pairs like A&T and G&C and
structure is double helix.
What is rDNA ?
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Recombinant DNA is genetically engineered DNA prepared
by transplanting or splicing genes from one species into
the cells of a host organism of a different species .
Thus the name recombinant DNA is also referred to as
“CHIMERA”.
PRINCIPLES
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Generation of DNA fragments and selection of the desired piece of
DNA.
Insertion of the selected DNA into a cloning vector(ex : plasmid) to
create a cloning vector or a chimeric DNA.
Introduction of the recombinant vectors into the host
cells(bacteria).
Multiplication and selection of clones containing the recombinant
molecules.
Expression of the gene to produce the desired product.
RESTRICTION ENDONUCLEASES
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These are the bacterial enzymes that can cut / split DNA at
specific sites.
Ex: E.coli R1, Hind 3.
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RECOGNITION SEQUENCE : It is the site where the DNA
is cut by a restriction endonuclease. Restriction
endonucleases can specifically recognize DNA with a
particular sequence of 4-8 nucleotides and cleave.
CLEAVAGE PATTERNS : The cut DNA fragments may have
sticky ends or blunt ends.
DNA LIGASES
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The cut DNA fragments are covalently joined together by
DNA ligases.
They form a phosphodiester bond between the phosphate
group of 5’- carbon of one deoxyribose with hydroxyl group
of 3’- carbon of another deoxyribose.
Ex : DNA polymerase 1- Synthesis DNA complementary to a
DNA template.
HOST CELLS
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Host cells are the living systems or cells in which the
carrier of recombinant DNA molecule or vector can be
propagated.
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Prokaryotic hosts : Escherichia coli, bacillus subtilis.
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Eukaryotic hosts : Yeast, sacchromyces cerevisiae.
VECTORS
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Vectors are DNA molecules, which can carry a foreign DNA
fragment to be cloned. They are self replicating in host cell.
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Plasmid
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Bacteriophages
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Cosmids
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Artificial chromosome vectors
(I) Human artificial chromosome (HAC)
(II) Yeast artificial chromosome
(III) Bacterial artificial chromosome(BAC)
PROCEDURES
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TRANSFORMATION : The first step in transformation is to select a
piece of DNA to be inserted into a vector.
The second step is to cut that piece of DNA with a restriction enzyme
and then ligase the DNA insert into the vector with DNA Ligase.
The insert contains a selectable marker which allows for identification of
recombinant molecules. An antibiotic marker is often used so a host cell
without a vector dies when exposed to a certain antibiotic, and the host
with the vector will live because it is resistant.
The vector is inserted into a host cell, in a process called transformation.
One example of a possible host cell is E. Coli. The host cells must be
specially prepared to take up the foreign DNA.
NON-BACTERIAL TRANSFORMATION :This is a process very
similar to transformation. The only difference between the two
is non-bacterial does not use bacteria such as E. Coli for the
host.
In microinjection, the DNA is injected directly into the nucleus
of the cell being transformed.
PHAGE INTRODUCTION :
Phage introduction is the process of transfection, which is
equivalent to transformation, except a phage is used instead of
bacteria. In vitro packagings of a vector is used.
This uses lambda or MI3 phages to produce phage plaques which
contain recombinants. The recombinants that are created can be
identified by differences in the recombinants and nonrecombinants using various selection methods.
CONJUGATION : It is a natural microbial recombinant
process during which two live bacteria come together join by
cytoplasmic bridges and transfer single stranded DNA(from
donor to recepient).
ELECTROPORATION METHOD : It is based on the
principle that high voltage electric pulses can induce cell
plasma membranes to fuse.
Electric shocks can also induce cellular uptake of exogenous
DNA from the suspending solution.
LIPOSOME MEDIATED GENE TRANSFER(LIPOFECTION)
On treatment of DNA fragment with liposomes, the DNA
pieces get encapsulated inside liposomes. These liposomes
can adhere to cell membranes and fuse with them to
transfer DNA fragments.
GENE CLONING
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GENE CLONING : The technique of making many copies of
a gene.
Generally genomic DNA is ideal for cloning, but the DNA
contains non coding sequences(introns) and repetitive
sequences.
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This complicates the cloning strategies, hence DNA is not
preferred.
Hence m-RNA is preferred.
m-RNA is preferred for the following reasons :
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m-RNA represents the actual genetic information being
expressed.
Selection and isolation of m-RNA are easy.
As introns are removed during processing m-RNA reflects
the coding sequence of the gene.
The synthesis of recombinant protein is much easier with
m –RNA cloning.
APPLICATIONS
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Essentially every area of biological research has been
affected by the use of rDNA technology. Protein
structure/function relationship studies and gene
expression and regulation research have been enormously
enhanced by this powerful tool.
Transgenic animals (into which DNA from another species
has been inserted) have been bred to expand the study of
human biochemical processes and diseases. Transgenic
mice that are highly susceptible to breast cancer or
Alzheimer's disease have furthered the understanding of
those diseases.
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Modern medicine is inextricably linked with rDNA
technology. Gene therapy replaces defective genes with
functional ones, delivered to the patient by way of a
suitable vector, usually a disabled virus. The first
moderately successful gene therapy was instituted to treat
an inborn immune deficiency disease (ADA deficiency)
caused by a defective enzyme, adenine deaminase.
Cancer research and treatments as well as some vaccine
development make use of rDNA technology. Attempts to
modify animals genetically in such a way that organs suitable
for transplant into humans may be harvested are now being
made.
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Agricultural uses of recombinant DNA technology are
expanding. Genetically engineered bacteria sprayed onto
strawberries protect the strawberries from freezing.
Genes that promote herbicide resistance are incorporated
into plants so that herbicides can be used for no-till
farming. Some plant species have been transformed by
rDNA containing genes that promote resistance to insects
and pathogens.
The industrial use of rDNA technology includes the
production of bleach-resistant enzymes that are used in
laundry detergents to degrade.
REFERENCES
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Molecular biology, David freifelder, second edition,
Narosa publishing house, pg.no 705-714.
Biochemistry , U . Satyanarayna , U.Chakrapani,
third edition, Books and allied publications, pg.no
578-615.