2014 notes

2. • Bio technology :
• Bio technology deals with the techniques of using live
organisms or enzymes to produce products and
processes useful to humans.
• The term Bio technology was coined by Karl Ereky in
1919.
• Principles of Biotechnology:
• Two imp techniques that used in modern
biotechnology are,
– Genetic engineering: Techniques to modification of
chemistry of genetic material (DNA and RNA) and to
introduce them into host organisms to change the
phenotypic character of the host.
– Maintenance of sterile: (microbial contamination-free): It is
the processes to enable growth of only the desired
microbe/eukaryotic cell in large quantities.

5. • The three basic steps involved in Genetic engineering
are,
1. Identification of DNA with desirable genes.
2. Introduction of indentified DNA in to the host.
3. Maintenance of introduced DNA in the host and
transfer of that DNA in to its progeny.
• The techniques of genetic engineering include
creation of recombinant DNA.
• r-DNA is used in gene cloning and gene transfer. It
helps to introduce desirable genes into the target
organism.
• It also helps to avoid undesired genes getting
multiplied along with the desired genes.

6. • The tools of recombinant DNA technology .
1. Restriction enzymes.
2. Polymerase enzymes.
3. DNA - Desired gene.
4. Ligases.
5. Vectors.
6. Host organism.
7. Bioreactors.

7. • Hamilton smith discovered and isolated
HIND II REN from Haemophilius influenzae in
1968. He received the Nobel Prize in
Physiology or Medicine in 1978.

8. • Restriction enzymes:
• REN are enzymes naturally occurring in bacteria as defensive enzyme. It
cuts and destroys bacteriophage DNA that infects bacterial cell.
• It recognize specific palindromic sequences to cut in DNA. Different types
of REN are identified and isolated for different palindromic sequence.
• The RENs are called molecular knives or molecular scissors as they cut DNA.
• Palindromic sequences: Palindromic sequences are ‘invert repeats’ which
have the same nucleotide sequences when read in 5‘to 3' on both the
strands.
• (As MALAYALAM.)

10. • The naming of restriction enzyme:
1. The first letter of the name comes from the genus of bacterium
from which they are isolated. .
2. Second letter from the species of the bacterium.
3. The third letter indicate the strain of the organism.
4. The roman numbers following the name indicate the order of
discovery.
• example: EcoRI comes from Escherichia coli RY 13
• RENs are Eco RI, Hind III, Sma I, Hae III, etc.,..
• The fragments of DNA produced are called restriction fragments. The
fragments of DNA may have ‘blunt ends’ or ‘staggered ends’
• (sticky ends).
• • Endonuclease : They breaks DNA double helix at any point except
the ends. They produce internal cuts called nick's or cleavage.
• • Exonuclease : They breaks or cuts the 5' or 3' ends of DNA molecule
i.e. they remove nucleotides from terminal ends of DNA.

11. • Desired gene: The functional or normal gene of
our interest taken from donor cell.
It is also known as foreign gene or trans
gene.
•Polymerase enzymes:
1. DNA Polymerase I.
2. DNA Polymerase II
3. DNA Polymerase III.

12. DNA ligase.
• The enzyme that joins the two sticky
ends of DNA is called DNA ligase. It is
used as molecular sitichers in genetic
engineering.
• DNA ligase was discovered by H G
Khorna.
• Dr. Hargobind Khorana was born on
9th January 1922 at Raipur, Punjab
(now in Pakistan).
• Died November 9, 2011 (aged
89)Concord, Massachusetts, U.S.
• In 1968, He was awarded the Nobel
Prize in Physiology or Medicine for the
interpretation of the genetic code and
its function in protein synthesis
1922 - 2011

13. • Vector: The carrier DNA that act as vehicle to carry
desired gene to the host cell is called vector.
• The imp vectors used in Genetic engineering are,
1. Plasmid.
2. Phages.
3. Plant virus.
4. Animal virus.
5. Cosmids.
6. Artificial chromosomes.
• Plasmid: The extra chromosomal self replicating
small circular DNA present in bacteria is called
plasmid.

14. • Cloning vectors:
• The plasmid and bacteriophages are used as cloning vectrors.
Bcoz it has the ability to replicate within bacterial cells
independentlly.
• Features of cloning vector:
• vector should contain three features for gene cloning. They
are
• 1) Ori site. 2) Cloning sites..3) selectable markers.
• Ori site: - Origin of replication
• The sequence of DNA where the replication starts is
called ori site.
• It helps the desired DNA linked with vector also replicates.
• Cloning sites:
• The vector contains the recognition site or palindromic for
the commonly used REN to insert desired gene.

17. • Host cell: The cell to which desired new gene is
introduced is called host cell. Any living cell can be
used as host cell.
• Commonly E.coli bacterial cell is used as host cell
in genetic engineering. Because,
1. It is a simple prokaryotic cell.
2. It is a non-pathogenic bacteria.
3. It can be cultured easily in laboratory condition.
4. It has very short life span.
5. It contains self replicating plasmid.
6. The plasmid of E.coli can be easily handled as
vector.

18. • The steps involved in recombinant DNA
terchnology.
1. Isolation of desired DNA .
2. fragmentation of DNA by restriction
endonucleases.
3. Isolation of a desired DNA fragment.
4. Ligation of DNA fragment in to vector.
5. Transferring the recombinant DNA in to host.
6. culturing the host cell and extraction of desired
product.

21. Extraction of DNA or isolation of gene:
1. The cells of organism that contains
desired gene are collected.
2. The DNA of these cells is extracted by
using refrigerated centrifuge technology.
3. The contents of the cell is treated with
enzymes like chitinase, lysozymes,
cellulase , ribonuclease and proteases.
4. Finally chilled ethnol is added to get
purified DNA.
5. The isolation of gene is done by shoot
gun method. In this specific REN is used
to cut and isolate desired gene. The
isolated gene contains two sticky ends.
6. The desired gene is isolated by using gel
electrophoresis.

22. • Gel Electrophoresis.
1. The fragments obtained after cutting with restriction
enzymes are separated by using gel electrophoresis.
2. Electric field is applied to the electrophoresis matrix
(commonly agarose gel).
3. The negatively charged DNA fragments move towards the
anode.
4. Fragments are separate according to their size due to the
sieving properties of agarose gel. Smaller the fragment move
faster than larger one.
5. Staining dye ethidium bromide followed by exposure to UV
radiations are used to visualise the DNA fragments.
6. DNA fragments are visible as bright orange coloured bands
in the agarose matrix.
7. These bands are cut from the agarose gel and extracted
from the gel piece (elution).
8. DNA fragments are purified and used in constructing
recombinant DNAs.

25. • Multiplication of gene: The isolated desired
gene is multiplied into millions of copies using
polymerase chain reaction.

26. • Ligation of desired gene in to vector.
• A suitable plasmid is selected. It is treated with the
same restriction enzyme to break and open the
plasmid at specific sites with sticky ends.
• Now the desired gene and the plasmid are mixed
and enzyme DNA Ligase is added .
• The desired gene gets incorporated in to the
plasmid .
• Transferring of recombinant plasmid in to the host
cell. (transformation)
• The recombinant plasmid is transfered in to the
host cell by cold calcium chloride method or by
micro-injection.

27. • The bacterial cell ( host cell) and r-DNA are
made to suspend in cold (5-6 0C ) calcium
chloride solution. After some interval of time,
the temp of solution is suddenly raised to 42
0C and again cooled.
• The increase in temperature increases the
pore size of bacterial membrane. Through this
pore r-DNA enters the bacterial cell .

28. • Micro-injection:
• Method of introducing r-DNA directly by injecting in
to the host cell using fine needle is called
microinjection.
• Biolastics or gene gun:
• Method of introducing recombinant DNA in to host
cell by bombarding high velocity microparticles of
gold or tungsten coated with DNA.

29. • Culturing the host cell and extraction of desired
product:
• The r-DNA is made to express under appropriate
conditions.
• The host cells are cultrured in a vessel called
Bioreactor.
• The desired protein is extracted and purified by
using different separation techniques called down
stream processing.

31. • Bio -reactor :
• Bio -reactor is an apparatus for culturing organisms like algae,
fungi, bacteria, animal cell or plant cells under controlled
conditions.
• It is used in industrial processes to produce pharmaceuticals,
vaccines, or antibodies.
• In bio -reactor raw materials are biologically converted in to
specific products.
• The most commonly used bioreactors are stirred tank reactor.
• It is cylindrical with curved base to facilitate the mixing of the
reactor contents. The stirrer facilitates even mixing. Air is bubbled
through the reactor.
• The reactor has,
1. an agitator or stirrer system,
2. an oxygen delivery system
3. a foam control system.
4. a temperature control system.
5. pH control system and
6. sampling ports.

34. • Downstream processing:
• Separation, purification of the products obtained
from recombinant DNA technique is called
downstream processing (DSP).
• The product has to be formulated with suitable
preservatives .
• Clinical trials and strict quality control testing is also
conducted before marketing of the product.

35. • Vectors used for cloning genes in plants.
• Ti plasmid: Tumour inducing Ti plasmid of
`Agrobactrium tumifaciens , used as cloning vector.
It is nonpathiogenic to the plants. It can be used to
deliver genes of our interest in to variety of plants.
• Vectors used for cloning genes in animals.
• Retroviruses. The trimmed non pathogenic
retrovirus are used to deliver desirable genes into
animal cells.