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Biotechnology

• Genetic engineering – direct manipulation


of genetic material for practical purposes
• Biotechnology – use of living organisms or
their components to make products for us
• Recombinant DNA – combining pieces of
DNA from different organisms
• Gene cloning – making copies of DNA
• Biotechnology deals with techniques of
using live organisms or enzymes from to
produce products process useful to
humans.

• EUROPEAN FEDRATION OF
BIOTECHNOLOGY defines biotechnology as
“The integration of natural science and
organisms, cells molecular analogues for
products and services “
• Among many the two core techniques that
enable birth of biotech are:
 GENETIC ENGINEERING –The technique to
alter the chemistry of genetic material to introduce them
into host organisms thus change their phenotype
 MAINTENANCE OF STERILE CONDITION
this enables the growth of only desired microbe in
large quantity for manufacture of products like
vaccines
This technique includes creation of
 Recombinant DNA
 Gene cloning
 Gene transfer
Which allows isolation of only desired genes
and characters
• A DNA sequence (e.g. a gene) can be
removed from a chromosome using special
enzymes

• Restriction enzymes are nucleases that cut


DNA at specific nucleotide sequences
• The are of two kinds exonulease and
endonuclease
• Exonulease remove nucleotide from end
of DNA whereas endonuclease make cuts
at specific positions with in DNA
ACTION OF RSTRICTION
ENDONUCLEASE
GEL ELECTROPHORESIS
• Four steps of gel electrophoresis
1.DNA mixtures are placed into wells at one end of
a slab of agarose gel

2.An electric current introduced through the gel


causes the negatively-charged DNA fragments to
migrate towards the positive electrode
3. Short DNA fragments move more easily
through the three-dimensional meshwork of
fibers between the gel
– Short DNA fragments migrate farther than long
DNA fragments so the mixture is separated into
bands of DNA of specific lengths
4. The invisible bands of DNA are made visible
using stains or DNA probes
• Recombinant DNA tech involes syeps like
 Isolation of genetic material
 Cutting of dna at specific location
 Ampification of gene using PCR
 Insertion of recombinant dna into host cell
 Obtaining the foreign gene product
ISOLATION OF GENTIC
MATERIAL
• The genetiv material is enclosed ina
membrane we have to break open to release
the genetic material. This can be
achived by treating cells with lysozyme
or cellulase or chitinase
• To seprate RNA from DNA it can be
treated with ribonulease.Protein can be
removed by protease
• Dna can be precipitated after addition
of chilled etanol
CUTTING DNA AT SPECIFIC
LOCATION
• Restriction enzyme digestion are
performed by incubating purified DNA
molecule with restriction enzyme at
optimal condition
• Agarose gel electrophores is employed to
check the progression of restriction
enzyme digestion
• Aftr the source and vector DNA are cut
they are mixed wiyh liase to prepare
recombinant DNA
Ampification of gene using PCR
• Four steps of a PCR cycle
1. Template strand separation
– The test tube is heated to 90-95oC to cause the
double stranded template DNA to separate into
single strands…

2. Binding of the primers


– The temperature is lowered to 50oC to allow the
primer DNA segments to bind to the targeted gene
sequences through hydrogen bonding…
Polymerase Chain Reaction

3. New DNA synthesis at targeted sequences


The temperature is raised to 70-72oC where the
heat-stable DNA polymerase synthesizes new
DNA of the sequences targeted by the primers…

4. Repetition of the cycle


The cycle is repeated automatically (by a
thermocycler machine) for 20-30 cycles,
producing up to 1 billion copies of the original
targeted DNA sequence
Polymerase Chain Reaction:
(a) One PCR Cycle

90 °C 50 °C 72 °C

DNA Polymerase Primer


DNA

Original Separate Primers & DNA


Double- DNA DNA synthesize
helix Strands polymerase
DNA bind
Polymerase Chain Reaction:
(b) Multiple PCR Cycles

2 copies 4 copies 8 copies

DNA
ragment
to be
amplified
Polymerase Chain Reaction

• Choice of primers determines which


sequences are amplified (copied)
• Forensic scientists focus on short
tandem repeats (STRs) found within
the human genome
Polymerase Chain Reaction

• STRs are repeated sequences of DNA within


the chromosomes that do not code for
proteins
• STRs vary greatly between different human
individuals
• A match of 10 different STRs between
suspect and crime scene DNA virtually
proves the suspect was at the crime scene
Inserting a recombinant DNA ina
host cell/organism
• There are many methods of introducing
the DNA into recipent like
 MICRO-INJECTION: in this method the
recombinant DNA is directly injected in the
nucleus of the animal cell
 GENE GUN: Plant cell are bombarded
with small particles of gold or tungsten
coated with DNA
• After makng the recipent cells compitent
they take up DNA present in their
surrounding. for eg
If a recombinant DNA bearing gene for
resistance to ampicillin is transferred to
Ecoli. The host cells become ampicillin
resistance cells. If send transformed cells
on agar plates containg ampicillin only
transformed cells grow
Obtaining the foreign gene product
• In almos all recombinant technology the ultimate aim is to
achive desired protein.
• If any protein encoding gene is expressed in heterogeneous
host its called a RECOMBINANT PROTEIN
• The cells harbouring cloned genes of interest may be ggrown
on small scale in lab and cutlure may be used to extract
protein
• To produce large quantity products BIOREACTORS are
used where large volume culture can be processed
• The most commnly used
bioreactors are stirring
type
 The are usually
cyclinders. Stirrer
facilitates even mixing
and oxygen avalability
through reactor
 It has foam control, temp
control, pH control, foam
control, oxygen delivery,
agitator systems
 It has a sampaling port
to check small volume of
culture periodically
DOWNSTREAM PROCESSING
• After compeletion of biosynthetic stage,the
producct is subjected to process like
separation and purification (downstream
process) before marketting.
• The product has to be formulated with
suitable preservatives and go through
clinical test in case of drugs
• Strict quality control test for the product
are also required
BIOTECHNOLOGICAL
APPLICATION IN AGRICULTURE
Genetically modified plannts have many
advantageslike
 These ccrops are more tolerant to abiotic stress (heat,
cold, drought)
 Reduce reliance on pesticides
 Reduced post harvest loss
 Increased effeciency of mineral usage
 Enhanced nutritional value
• Among many genetically modifeied cops
developed by man BT cotton dnd pest
resistant crops are the one we are going
to discuss