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Definition
• Biotechnology is the
integration of natural
science and organisms,
cells, parts thereof and
molecular analogues for
products and services.
Oldest form of biotechnology
Application of
fermentation in
production of wine and
other alcoholic
beverages is also a
biotechnological
technique
But with time biotechnology gradually became more
sophisticated.
Biotechnology
DNA manipulation
Tissue culture
Protoplast fusion
Cell catalysis
Immobilized enzymes
Protein engineering
Biotechnology led to production of many products and provides many services for human welfare.
Dragon Fly
There were 180,000 people employed by U.S. biotechnology companies in 2006.
There are more than 400 biotech drug products and vaccines currently in clinical trials targeting more than 200 diseases, including various cancers, Alzheimer’s disease, heart disease, diabetes, multiple sclerosis, AIDS and arthritis.
The biotechnology industry has mushroomed since 1992, with U.S. health care biotech revenues from publicly traded companies rising from $8 billion in 1992 to $58.8 billion in 2006.
In-Vitro Fertilization
Also called as
Test tube baby
Plant tissue culture
DNA vaccines
The recombinant DNA technique was
first proposed by
Peter Lobann A. Dale Kaiser
The present day rDNA technology flourished after the work of
Salmonella typhimurium
E. coli
They successfully linked a gene
coding for antibiotic resistance with
a native plasmid of Salmonella
typhimurium with the vector plasmid
and then cloning it in E.coli.
Plasmid
Gene coding for
antibiotic resistance
Vector
Cloning
• Technique of manipulating the genome of a cell
or organism so as to change the phenotype
desirably.
What is recombinant DNA ?
Seedless guava Calorie free sugar
Isolating genomic
DNA
Fragmenting this DNA Screening
the fragments
Insertion of DNA in a vector
Introducing in Host
Culturing the cells
Transformation of host cell
Isolating genomic
DNA
Isolating
genomic DNA
from the donor.
Fragmenting this DNA
Fragmenting
this DNA using
molecular
scissors.
Insertion of DNA in a
vector
Screening the
fragments
Screening the
fragments for a
“desired gene”.
Inserting the
fragments with the
desired gene in a
„cloning vector‟.
Introducing in Host
Culturing the cells
Transformation of host cell
Introducing the recombinant
vector into a competent host
cell
Culturing these cells to obtain
multiple copies or clones of
desired DNA fragments
Using these copies to
transform suitable host cells
so as to express the desired
gene.
Example: Production of Insulin
Tools used in recombinant DNA technology
• Enzymes
• Vectors
Tools used in recombinant DNA technology
• Enzymes
Act as biological scissors.
Most commonly used are:
Restriction endonuclease
DNA ligase
DNA polymerase
Alkaline phosphatases
Tools used in recombinant DNA technology
• Vectors
Low molecular weight DNA molecules.
Transfer genetic material into another cell.
Capable of multiplying independently.
Vector
Vector
Bacteriophage DNA
Artificial DNA
Cosmid
Plasmid
Insertion of vector in target cell is called
• Bacterial cells – Transformation
• Eukaryotic cells – Transfection
• Viruses - Transduction
Insertion of vector in target cell
Vectors used:
• Bacteria- plasmids, cosmid, lambda phage
• Insects- baculoviruses
• Plants- Ti plasmid
• Yeast cells- YAC (yeast artificial chromosome)
HOST DONORDNA
Fragmented by Restriction Endonuclease
DNA strands with sticky ends
Sticky ends base pair with complementary sticky ends
DNA ligase links them to form rDNA
Cloned In vivoIn vitro
Prokaryotic or eukaryotic cell, mammalian tissue culture cell
DNA
Polymerase chain reaction (PCR)
Some examples of therapeutic products
made by recombinant DNA techniques
¶ Blood Proteins: Erythropoietin, Factors VII, VIII, IX; Tissue
plasminogen activator; Urokinase.
¶ Human Hormones: Epidermal growth factor; Follicle
stimulating hormone, Insulin.
¶ Immune Modulators: α Interferon, β Interferon; Colony
stimulating hormone; Lysozyme; Tumor Necrosis factor.
¶ Vaccines: Cytomegalovirus; Hepatitis B; Measles; Rabies
Transposons
• Transposons are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell.
• Also called „Jumping genes‟.
• 1st transposons were discovered by
Barbara McClintock
in Zea mays (maize)
Types of transposons
• According to their mechanism they are classified as:
Retrotransposons
• Follows method of “Copy and Paste”.
• Copy in two stages.
DNA DNARNA
Reverse Transcription
Transcription
DNA transposons
• Follows the method of “Cut and Paste”.
• Do not involve RNA intermediate.
Enzyme Transposase
Cuts out transposon
Ligates in new position
Plasmid
• Plasmids are small, extra chromosomal, double
stranded, circular forms of DNA that replicate
autonomously.
• The term was introduced by in 1952.
Joshua Lederberg
Plasmid
• Found in bacterial, yeast and occasionally in
plants and animal cells.
• Transferable genetic elements or
‘Replicons’.
• Size- 1 to 1000 kilo bp.
• Related to metabolic activity.
• Allows bacteria to reproduce under
unfavorable conditions.
Nomenclature
Lower case P (p)
First letters of researchers name or place where
it was discovered.
Numerical numbers given by workers.
Plasmid
Plasmid
Eg. Plasmid pBR 322
BR is for Bolivar and Rodriguez, who designated it as 322
Plasmid Eg. Plasmid pUC 19
UC stands for University of California
Plasmid- Cosmids
• Cosmids are plasmids with cos sequence.
• They are able to accommodate long DNA fragments
that plasmids can’t.
A bacteriophage is a virus that infects bacteria.
Virulent portion is deleted.
Genetic material can be
ssRNA, dsRNA, ssDNA,
dsDNA.
For Single genes- Plasmids are used
For Large pieces of DNA- Bacteriophages
48.5 kb in length.
Cos sites of 12 bp at the ends.
Cohesive ends allow circularizing DNA in host.
(1) Phage attaches to a specific host bacterium.
(2) Injects its DNA, (3) Disrupting the bacterial genome
and killing the bacterium, and (4) Taking over the bacterial DNA and
protein synthesis machinery to make phage parts.
(5) The process culminates with the assembly of new phage, and
(6) The lysis of the bacterial cell wall to release a hundred new copies of the input phage into the environment.
RESTRICTION FRAGMENTS
A restriction fragment is a DNA fragment resulting
from the cutting of a DNA strand by the restriction
enzyme.
Process is called restriction.
RESTRICTION FRAGMENTS
Steward Linn along with Werner Arber in 1963
isolated two enzymes.
One of them is Restriction Endonuclease.
Restriction Endonuclease can cut DNA.
Restriction Endonuclease are basic requirement
for gene cloning or rDNA technology.
RESTRICTION FRAGMENTS
Nucleases
EndonucleaseExonuclease
They remove
nucleotides
from the ends
of the DNA
They make cuts
at specific
positions within
the DNA
TYPES OF REN
REN
Type I Type II Type III
Mostly used in rDNA technology.
More than 350 types of type II
endonucleases with recognition sites are
known.
Can be used to identify and cleave within
specific DNA.
NOMENCLATURE OF REN
First letter- genus name of bacteria (in italics).
Next- first two letters of the species name (in
italics).
Next- strain of the organism.
Roman number- order of discovery.
Eg. - EcoR I
E- Escherichia, co- coli, R-strain Ry 13,
I- first endonuclease to be discovered.
Eg.- Hind III
H- Haemophilus, in- influenzae, d- strain Rd,
III- third endonuclease to be discovered.
NOMENCLATURE OF REN
RECOGNITION SEQUENCE (RESTRICTION SITES)
It is the site/ sequence where REN cuts the DNA.
Sequence of 4-8 nucleotides.
Most restriction sites are Palindromes.
In DNA, palindrome is a sequence of base pairs
that reads the same on the two strands when
orientation of reading is kept same.
CLEAVAGE PATTERNS OF REN
REN recognizes the
restriction site.
Cleave the DNA by
hydrolyzing
Phosphodiester bonds.
Isolate a particular gene.
Single stranded ends
called sticky ends.
These sticky ends can
form hydrogen bonded
base pairs with
complementary sticky
ends or any other cleaved
DNA.
CLEAVAGE PATTERNS OF REN
Restriction fragments
yield a band pattern
characteristic of the
original DNA molecule &
restriction enzyme used.
Gel
electrophoresis
Bands
PREPARING AND CLONING A DNA LIBRARY
Collection of DNA fragments from a particular species that
is stored and propagated in a population of micro
organisms through molecular cloning.
GENOMIC LIBRARY
Collection of all clones of DNA fragments of complete
genome of an organism.
All DNA fragments are cloned and stored as location of
desired gene is not known.
Screening of DNA fragments can be done by
Complementation Or by using Probes.
Entire genome isolated
Cut into fragments by REN
Fragments inserted in Vector
Recombinant vectors are transferred into suitable organism
Transferred organisms are cultured and stored
Construction of Genomic Library.
CDNA LIBRARY
cDNA is Complementary DNA.
Produced using Teminism i.e. Reverse Transcriptase.
Constructed for eukaryotes.
cDNA is made from mRNAMature mRNA
StartAAAAAAA
Stop
TTTTTTT Add polyT primer, nucleotides, and Reverse Transcriptase
TTTTTTT AAAAAAA
TTTTTTT
DNA/RNA
RNA removed (by NaOH) and second strand synthesized
Complementary DNA cDNA
Gene Amplification (PCR)
It is obtaining multiple copies of a known DNA
sequences that contain a gene.
Done artificially by using PCR (Polymerase Chain
Reaction)
PCR (Polymerase Chain Reaction)
Developed by in 1983.
In Vitro technique.
Scientific technique to generate billions of copies of a
particular DNA sequence in a short time.
Kary Mullis
PCR Machine
Requirements for PCR technique
DNA segment Primers
dNTPsThermostable
DNA polymerase
PCR
A DNA segment
100-35,000 bp in
length to be
amplified.
Primers-forward and
reverse, are
synthetic
oligonucleotides and
complementary to
the desired DNA
segmentFour types of
deoxyribonucleotid
es i.e. dCTP, dGTP,
dTTP, dATP
Enzyme that can
withstand upto 94°
C.
Steps of PCR technique
The double strand melts open to single
stranded DNA, all enzymatic reactions stop
(for example : the extension from a previous
cycle).
Ionic bonds are constantly formed and
broken between the single stranded primer
and the single stranded template. Once
there are a few bases built in, the ionic bond
is so strong between the template and the
primer, that it does not break anymore.
The bases (complementary to the template)
are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3',
reading the template from 3' to 5' side,
bases are added complementary to the
template)
The exponential amplification of the gene in
PCR.
Application of biotechnology in
agriculture- Bt crops
Bacillus thuringiensis
• Soil bacterium.
• Produces a protein that has insecticidal properties.
• Traditionally used as spray.
Mechanism of Bt
• Bt produces Bt toxins which are inactive protoxins.
• When an insect ingests it, inactive protoxin gets converted into active form due to alkaline pH of the insect’s gut.
• This led to swelling of gut and ultimately death of insect
Bt (in inactive form) sprayed on Crops
Eaten by insect
Toxin gets activated by alkaline pH of insect’s gut
Swelling of gut of insect
Death of insect
• Cry gene in Bt produces inactive protoxins.
Crop plants are now engineered to express Bt toxin.
Bt crops are now commercially available.
For Eg.
Bt Rice Bt Cotton Bt Tomato
Bt Brinjal Bt Soybean Bt Potato
Bt Corn
Agrobacterium tumefaciens
• Soil bacterium.
• Causes crown gall tumors in dicotyledonous plants.
• T DNA (gall producing gene) occurs in Ti plasmid.
• Ti plasmid is used as vector for higher plants.
• Many genetically modified plants are produced using A. tumifaciens.
Tumor
Mechanism
Ti Plasmid
• Desirable genes such as Cry gene an Nif gene is cloned inside A. tumifaciens and then transferred into another plant.
Nif Gene isolated from Rhizobium
Examples
1. Flavr savr tomato
Longer shelf life.
Antisense DNA is introduced that retards ripening
2. Golden Rice
Greater pro vitamin A content.
Genetically engineered.
Bio-Safety Issues Biosafety
issues
Impact on Agriculture
Ethical issues
Impact on human health
and environment
Genetically
modified
organisms
Genetic modification of organisms
can lead to Contamination of gene pools.
Consumption may lead to allergies.
Hazardous microbes may escape
laboratory
Therefore manipulation of organisms
needed regulation
Genetic Engineering Approval
Committee
In India, GEAC takes decision regarding validity of GM
research and introduction of GM products.
Biopiracy The patenting of plants, genes, and other biological
products that are indigenous to another country
Developed countries patent the knowledge and
resources of underdeveloped countries and enjoy
immense profits.
Biopatent
A patent is granted
by the government
to the inventor for
biological entities,
processes and
products.
Case Study
Texmati was derived
by crossing Indian
Basmati rice with a
semi dwarf variety.
A Texas based
company got patent
on rights of basmati.Indian Basmati rice
Texmati rice
Some other Examples
Turmeric Neem Margosa
What can be done?
Genetic Literacy Movement in Schools and Colleges on rapid developments in Molecular Genetics
What will it do?
Better understanding of opportunities and
risks of rDNA technology.
Promote safe and responsible use of tools
of genetic engineering.
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