Genetic engineering and Biotechnology
“Or how I stopped worrying and
learned to love the sheep.”
polymerase chain reaction (PCR)
scientific technique in
molecular biology to
amplify a single or a few copies of a piece of
DNA across several orders of magnitude,
generating thousands to millions of copies of
a particular DNA sequence.
The method relies on
consisting of cycles of repeated heating and
cooling of the reaction for
DNA melting and
replication of the DNA.
POLYMERASE CHAIN REACTION
Used in research
Study a particular sequence
Need of identical copies
method of cloning
lots of time and work
It uses enzymes to replicate DNA
Enzyme is isolated from bacteria and yeast.
developed in 1983
Principle of PCR
Desired DNA is heated
break the Hydrogen bond
Two strand separate
Primes are added to start Replication
Mixture is cooled.
Primers bind to the original to ssDNA.
Nucleotides and thermostable DNA
Nucleotides will bond with exposed bases of the
The original strand has formed a new CDNA.
Cycle is repeated
Gel electrophoresis is a
technique used to
separate fragments of
Separates fragments as a
function of size and
Most types use
to separate fragments.
is a porous gel.
It can allow the passage of
however, larger molecules
move more slowly through
it since they cannot
squeeze through the pores
as easily as smaller
An agarose gel is casted with several
holes called wells at one end.
The gel is placed in an electrophoresis
box which is filled with an electrolyte
Samples of digested DNA are placed
in the wells
Electrical leads are attached to the
ends of the box creating an electrical
potential across the apparatus.
Because DNA has a negative electrical
charge. It is "pulled" towards the
positive side of the apparatus.
Also, since the smaller molecules
travel faster through the agarose. Over
time this separates the various sized
fragments of DNA.
The gel is then removed and stained
for DNA. This results in a gel which
shows several bands of stained DNA.
DNA profiling is also known as DNA fingerprinting
DNA is now a powerful tool in identification.
Based on the fact that the amount of "junk DNA" differs uniquely between
Structural genes are often separated by large regions of repeating base
The number of these repeats is unique to an individual.
Therefore when DNA from a person is cut with a restriction enzyme, the
length of the fragments will be unique to an individual.
DNA Fingerprinting Contd…
This will therefore
produce a unique
following a gel
This test is highly
accurate, and the
probability of another
an identical banding
pattern is estimated as
Small amounts of DNA
Amplify the DNA
Two strands are separated
endonucleases used to
Sections will differ in size and charge
Separated by Gel electrophoresis
Pattern of stripes and bands determined by the
sequence of the bases
. Paternity profiling
each band shown on the DNA Profile of a child
must correspond with a band of the father or
b. Forensic investigation
Compare DNA from the suspect with DNA from
the crime scene sample (blood, hairs, semen
c. Using relative’s DNA to determine the identity
of a victim
to determine the identity of the remains of dead
For Eg., Tsar of Russia and his family was shot
during the Russian revolution and bodies were
shown to prove it.
By taking blood samples of distant relatives of
DNA patterns could be established
Bodies were likely to be the Romanov family
for some ideas of
problems when using DNA profiling as
Human genome project
commitment undertaken by the scientific
community across the world.
International Human Genome Organisation
is an excellent example of how
collaboration of scientists across the world
can benefit all of us.
To determine the locations and structure of
all genes in the human chromosomes
Data was pooled
Suggested in 1985
base pairs) in human
In 2003 the sequencing of the human DNA was
Mapping of genes
listing and finding the locus
of each human gene
Outcomes of having sequenced the
entire human Genome
An improved understanding of many genetic
The production of medicines (based on DNA
sequences) to cure and/ or genetic engg. To
remove the gene which causes the diseases
To determine fully which genetic diseases any
individual is prone to ( genetic screening
leading to preventive medicine)
Research into a particular disease can focus on
Provide more info about evolutionary paths by
comparing similarities and differences in genes
Info is valuable BUT it could be abused.
insurance companies, prospective employer
and society faces the challenge of coming to
terms with the ethical issues
Deliberate manipulatipn of genetic material.
This is possible due to
Universal genetic code
Transfer the genetic material from one
species to another
Introduce human gene for making insulin into
The bacterium produces human protein
Requires following elements
to carry the gene into the host
A host cell
Restriction enzymes are compounds first
isolated in the 1970's
They function by selectively cutting DNA at
These cuts usually occur
in the following forms.
The cut can be made
straight across a base
pair sequence resulting in
The cut can be made in
an offset manner leaving
exposed sequences are
The presence of
sticky ends allows
segments of DNA to
be joined together.
Since DNA strands
which have been cut
by the same
can easily bond
to base pairing
Gene Splicing contd..
This allows for genes to be
"cut & pasted" between
organisms. This can be seen
with production of human
The DNA sequence of insulin
is identified and cut out using a
A plasmid from
removed and cut open using
the same restriction enzyme
Since both fragments have
complimentary sticky ends and
the gene for human insulin is
integrated into the plasmid
The plasmid is then reinserted
into a bacterial cell. This cell
will produce insulin and is
cultured. Human insulin can
now be extracted and provided
GM crops and
Genetically manipulated organisms
Called transgenic organisms
first GM food was sold commercially.
Tomato was altered
stay fresh longer
Gene was introduced to block the enzyme
which cause rotting
No longer available
G M Maize.
from Bacillus thuringiensis
Incorporated into maize
Plants produce a toxin that makes them
resistant to insects
Bt crops are grown in US
European corn borer, ECB found also in US
The ECB through stems and leaves of the corn
plant and will damage vascular bundles
disrupt the transport of water and nutrients
through the plant.
It can also weaken the stems and leaves so
that the plant or leaves may break
Bt corn is already in commercial use.
benefits of Bt corn
The damage caused by the ECB is much
Bt corn is slightly more expensive, but the
difference is less than one extra application
Bt corn needs to be checked often for
signs of ECB
less checking needed for Bt
Less insecticide needed means less impact on
the environment and lower health risks for the
•Seems to reduce the infection with fungus so
mycotoxin (poisons produced by fungi) levels
Mycotoxins are difficult to remove by
cooking/freezing and may go into the food chain
and be found in meat of animals which ate the
infected corn. Mycotoxins can be a hazard to
human and animal health.
harmful effects of bt corn
Will also kill some other insects (though
many are not affected).
Insects may develop resistance to Bt toxin
because they are exposed to it all the time
Resistant insects also make Bt spray
useless as insecticide (Bt spray is
considered to be relatively safe for humans
and the environment).
It is difficult to prevent pollen (with the Bt
gene) from travelling outside the field where
the Bt corn is grown
it may fertilise non
Bt corn e.g.
organically grown corn which can then
no longer be sold as organic corn.
it may fertilise wild relatives and make
them more resistant to insects and have
them dominate the niche they live in.
This would result in loss of biodiversity.
to study the disease polio
Possible treatment and prevention.
Rice is major part of their diet
Suffer from Vit A deficiency
lead to blindness
Rice store Kit A in leaves but not in rice grains
By adding genes from daffodills and from
Plant stores a precursor of Vit A in the grains
New kind of rice is now produced
One gene from maize and bacterium
20 times more of beta carotene compared of
valuable source of Vit A
opposed by environmentalist, and anti
Group of genetically identical organisms or
group of cells derived from a single cell
Using differentiated cells is mostly somatic cells
Cloning: What it is
is the process of making a
genetically identical organism
through nonsexual means.
It has been used for many years to
produce plants (even growing a plant
from a cutting is a type of cloning).
Animal cloning has been the subject
of scientific experiments for years,
but garnered little attention until the
birth of the first cloned mammal in
1997, a sheep named
Since Dolly, several scientists have
cloned other animals, including cows
The recent success in cloning
animals has sparked fierce debates
among scientists, politicians and the
general public about the use and
morality of cloning plants, animals
and possibly humans
Dolly, the first mammal clone
Dolly: A Mammal Clone
In 1997, cloning was revolutionized
and his colleagues at
the Roslin Institute in Edinburgh,
Scotland, successfully cloned a sheep
. Dolly was the first cloned
Wilmut and his colleagues transplanted
a nucleus from a mammary gland cell
of a Finn Dorsett sheep into the
enucleated egg of a Scottish blackface
egg combination was
to fuse the
two and to stimulate cell division.
The new cell divided and was placed in
the uterus of a blackface ewe to
develop. Dolly was born months later.
Clone a MIMI mouse
Creates a new cloning
Dolly the sheep
Dolly is known as SCNT
it is theoretically possible to apply the same
technique to cloning other species.
Horses are an example of a species cloned
successfully, but attempts with several other
species have been less successfull.eg
Eg., mare and her cloned foal
Involves stem cell research
Human embryos are produced and allowed to
grow for few days into small of ball of cells
These cells are not specialised but when SCNT
is used the cells can grow into any different
Other sources of stem cells from umbilical cord
or cells from aborted fetuses.
Aims for cell therapy
Bone marrow transplants, skin cells for
Grow new corneas
Ethical issues of Therapeutic
cloning in humans
Arguments in favor of therapeutic cloning
the ability to cure serious diseases with
currently leukemia and
in the future possibly cancer and
Some of the concerns raised about
therapeutic cloning relate
fears of it leading to reproductive cloning
use of embryonic stem cells involves the
creation and destruction of human embryos
(although it is possible to use embryos left over
from IVF treatment which would be destroyed
embryonic stem cells are capable of many
divisions and may turn into tumors.
Ethical aspects of cloning are difficult to discuss
since a lot of the benefits are currently not yet
realised. They are potential benef