Chap_12_Biotechnology.ppt

Biotechnology is a branch of biology which
deals with techniques of using live organisms,
enzymes or biological processes to produce
products & provide services for human welfare.
Food preservation
 Following are the fields in which
biotechnology is used
 Biotechnology is used by man from ages
Bread making
Wine making
Fermentation
Manufacture of soaps.
INTRODUCTION

Apart from this Test tube baby, DNA vaccine, PTC
are few more advanced applications
 In modern days apart from traditional use, it also
provides advanced tools & techniques like Recombinant
DNA Technology, Protoplast fusion, Cell catalysis,
Protein engineering, immobilized enzymes etc.
It also has wide applications in the fields of
Agriculture, Medicines, Chemical & Pharmaceutical
industries.
INTRODUCTION contd…..

“Biotechnology is integration of natural sciences & organisms, cells, parts
thereof & molecular analogues for products & services.”
The new definition given by EFB (The European Federation
of Biotechnology) is as follows;
We will be dealing with
Recombinant DNA technology
Transposons, Plasmids & Bacteriophages
Restriction Fragments
Preparing & Cloning a DNA Library
Application in Agriculture.
DEFINATION

Gene transfer from unrelated donors can also be done
successfully.
Recombinant DNA technology is the technique of
manipulating the genome of a cell or organism so as to
change the phenotype desirably
It was first proposed by Peter Lobban, it flourished
after Stanley Cohen & Herbert Boyer linked gene
coding for antibiotic resistance.
RECOMBINANT DNA TECHNOLOGY

Fragmentation of DNA by molecular scissors
(Enzymes)
Isolation of genomic DNA (DNA from Donor)
Screening for desired gene
STEPS in rDNA TECHNOLOGY
Inserting the fragment (desired gene) into Vector.
Vectors are plasmids, cosmids, phage DNA
RECOMBINANT DNA TECHNOLOGY contd…
Introduction of recombinant vector in host
Culturing the host to obtain many copies.
Using these copies for transforming host to express
desired gene

TOOLS USED in rDNA TECHNOLOGY
Enzymes like restriction endonucleases, DNA ligases,
Reverse transcriptase, DNA polymerases, alkaline
phosphotases etc.
Restriction endonucleases are used to cut the DNA at
specific sites hence are termed as biological / molecular /
chemical Knives / scissors / scalpels.
(I) ENZYMES
Restriction endonucleases cuts the DNA at specific
sites and form restriction fragments.(process -- restriction)
Steward Linn & Werner Arber isolated two enzymes.

(I) ENZYMES
Restriction enzymes belong to a larger class of
nucleases (exonucleases – cut at the ends of DNA & endonucleases—cuts
at specific positions within the DNA)
There are 3 major types type I, type II & type III, type
II are used in rDNA technology (can be used invitro, cleave within
specific DNA sequences 4-8 nucleotides).
More than 350 type II endonucleases with 100
different recognition sequences are known.

(I) ENZYMES -- Nomenclature
Following are the names of few restriction
endonucleases EcoR I, Hind III.
EcoR I – E – Escherichia co – coli strain R -- Ry 13
& I indicates first endonuclease.
Hind III – H Haemophilus in influenza strain Rd – d
III indicates third endonuclease.
The first letter (italics) indicates genus, followed by
two letters (italics) indicates species, then comes strain &
finally a Roman numeral indicating order of discovery.

(I) ENZYMES – Recognition sites/sequences
Recognition sites/sequences are those sites where the
DNA is cut by the enzyme, the enzymes specifically
recognizes DNA with a particular sequence (4-8 nucleotides
in length) & cleaves.
Each Restriction endonuclease is highly specific, cuts
the DNA at specific points at same time, most restriction
sites are palindromes (mirror image eg. MALAYALAM).
G A A T T C
G
A A
T T
C
T T
C
T
C
A G
G A
A
A
T
G
C
T
A
5”
5”
3”
3”

(I) ENZYMES – Cleavage patterns
Restriction endonucleases cleave the DNA by
hydrolysing the phosphodiester linkage (retains symmetry).
The double stranded fragment has sticky ends due to
restriction endonuclease (especially type II).
Mg++ ions are required for cleavage, many cuts are
made due to repetition of sequences.
Fragments are analyzed by techniques like gel
electrophoresis.
Fragments yield band pattern characteristic of the
original DNA.

They are usually plasmids, cosmids, BAC’s, YAC’s etc.
They are low molecular weight DNA molecules
They must have replication origin (a sequence elements
recognized by host cell’s replication machinery), so that
they can replicate.
(II) VECTORS
They should also have restriction endonuclease
recognition sites, & some marker genes which express in
the host.

PROCESS of rDNA TECHNOLOGY
DNA from both the sources is fragmented
Fragments show blunt/sticky ends.
DNA ligase covalently
links the strands into a
molecule of recombinant
DNA
This recombinant DNA/chimeric DNA is replicated
by PCR (in vitro) & through unicellular prokaryotes (in
vivo).
Bacterial
DNA
Blunt ends
Sticky ends

Therauptic products of rDNA TECHNOLOGY
Blood proteins – Erythropoietein, Factors VII, VIII,
IX, Tissue Plasminogen activator – Urokinase.
Human hormones – Epidermal growth factors, FSH,
Insulin
Nerve growth factor – Relaxin & Somatotrpoin
Immune Modulators – α & β interferons
Colony stimulating factor – Lysozyme, Tumor
necrosis factor.
Vaccines– Cytomegalovirus, Hepatitis B, Measles,
Rabies.

PLASMIDS
 Plasmids are extra-chromosomal, autonomously, self-
replicating mini-chromosomes found in prokaryotes
(bacteria).
 They range from 1 to more than 1000 kbp’s.
 They carry genes related to metabolic activity, also
allow the carrier to survive & reproduce in
unfavorable conditions.
 Cosmids are special plasmids having fragments of
lambda phage vectors.
 Cosmids are made because plasmids cannot
accommodate long DNA fragments.
pBR 322 – Bolivar & Rodriguez, pUC – University of California
 Nomenclature of Plasmids is as follows

BACTERIOPHAGE
Bacteriophage is a virus that infects bacteria
commonly termed as phage.
They consists of a capsid (protein layer) & genetic
material.
Genetic material can be ssRNA, dsRNA, ssDNA,
dsDNA in circular/linear arrangement.
Bacteriophages are better vectors than Plasmids as
they are efficient in cloning larger pieces of DNA.
Commonly used phages are M13, Lambda phage
Portion of phage DNA required for packing is retained
& then desired fragment is loaded.
Cosmids contain fragments of lambda phage vectors.

BACTERIOPHAGE CONTD…
DNA of lambda phage is 48.5 kb in length. It has
cohesive sites of 12 bp at ends, which allows
circularisation of DNA in host cell.
Phage DNA is replaced with desired gene (20kbp), the
recombinant DNA is packed in viral particles.
Once inside the host cell the rDNA starts replicating,
growth takes place by lysis, released viruses infect
other surrounding cells.
Cloned DNA can be obtained from the plagues of
lysed bacterial cells.

REPLICATION OF BACTERIOPHAGES
It takes places as follows
Attachment
Penetration
Synthesis of Proteins & Nucleic acids
Phages attach to specific receptors by random
encounters.
Tail fibres brings the base plate closer to the surface,
tail contracts, injects genetic material, protein coat
remains outside (ghost).
Normal synthesis of proteins & nucleic acids is
disrupted, and forced to manufacture viral DNA &
proteins, parts of new virions

REPLICATION OF BACTERIOPHAGES
Virion assembly
Release of virions
Base plates are assembled first with tails first, the
heads/capsids are constructed separately, then joined.
DNA is packed within the heads, the whole process
takes about 15 minutes.
They are released by lysis of cell, due to endolysin
(enzyme), infection spreads.

TRANSPOSONS
 Transposons/Jumping genes are sequences of DNA
that can move or transpose themselves to new
positions within genome of a single cell.
 They are of two types Retrotransposons & DNA
transposons
 Retrotransposons copy in two stages, from DNA to
RNA by transcription & then from RNA back to
DNA by reverse transcription.
 DNA transposons do not involve RNA , transposase
make a cut forms sticky end, cuts out the transposon
& ligates in new position.

DNA LIBRARY
Preparation & Cloning
cDNA library
Genomic library
Library is a collection of DNA fragments from a
particular species that is stored & propagated in a
population of microorganisms through cloning.
Genomic library is collection of all clones of DNA
fragments that represent complete genome of an
organism
It is complementary DNA /cDNA produced using
mRNA by the action of reverse transcriptase. The
process is called Teminism.

DNA LIBRARY CONTD….
As complete genomic DNA is cloned hence desired
gene can be screened by complementation or by using
probes
Each fragment is then inserted in cloning vectors.
Recombinant vectors are transferred to host.
Transformed host are cultured to produce clones &
stored
Genomic library
Entire genome is isolated & cut into fragments.

Thus a library having such cDNA for every structural
& functional protein can be constructed.
DNA LIBRARY CONTD….
In eukaryotes there are coding and noncoding
segments in DNA(non-coding segments are not required as they are not genes).
mRNA carrying only transcribed coding sequences
form proteins.
Such mRNA is collected from various cells, organs
etc at different time, phases (lifecycle) & converted in to
cDNA.
cDNA library
Used in interferons, insulin etc.

GENE AMPLIFICATION
PCR – Polymerase Chain Reaction
Obtaining multiple copies of known DNA is called as
gene amplification.
PCR (in-vitro) is artificial means of gene amplification.
Developed by Kary Mullis in 1983 (Nobel -- 1993)
Requirements
DNA segment to be amplified
Primers Deoxyribonucleotides
DNA polymerase (Tag polymerase)

GENE AMPLIFICATION
Steps in PCR
Heat Denaturation
Heating DNA at 91oC, breaks H2 bonds, makes
ssDNA. (if G & C pairing is more,higher temperature is required)
Pairing of primers to ssDNA, temperature -- 55oC.
Annealing
Tag polymerase adds dNTP’s behind primer on ss
DNA at 72oC.
Polymerisation

GENE AMPLIFICATION
Each cycle takes about 3-5 minutes, the newly
generated DNA itself can be used as template.
Thus it sets a chain reaction and results in exponential
amplification
Heat denaturation, annealing and polymerisation
complete one cycle.

APPLICATION IN AGRICULTURE
Use of Chemical fertilizers, Biological fertilizers &
GMO’s.
Of these options chemical fertilizers & biological
fertilizers have there own drawbacks, limitations
hence use of GMO’s becomes inevitable.
Increase in the food production is essential, it can be
brought about by following means
With transgenic plants we can design plants with
properties like disease resistance, insect resistance,
better photosynthetic efficiency, N2 fixation,
improved storage proteins, high vitamin content.

Bt toxin occurs as inactive protein, after ingestion due
to alkaline pH of the gut the inactive protein becmoes
active.
Due to this the gut swells resulting in the death of the
insect.
Bacillus thuringiensis is a soil bacterium with
insecticidal properties (Bt toxin protein).
Crop plants are engineered by rDNA technology to
contain & express gene (cry gene) for Bt toxin.
Through Bacillus thuringiensis

Tumours are induced due to Ti plasmid which has
ability to insert itself into the genome of the host
plant.
Cry gene/Nif gene can be cloned and introduced in
other crop plants through A. tumefaciens.
Agrobacterium tumefaciens is a soil bacterium
inducing tumours.
Through Agrobacterium tumefaciens

Flavr Savr tomato retards ripening by producing less
amount of Polygalactouranase enzyme (cell wall degrading enzyme)
giving more shelf life.
BIOSAFETY ISSUES
Biopatent is the right granted by the government to
the inventor for biological entities.
Biopiracy is the biopatenting of the bioresource of the
other nation without proper permission of the
concerned nation or unlawful exploitation and use of
bioresource without giving proper compensation.

Chap_12_Biotechnology.ppt

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Chap_12_Biotechnology.ppt