Oct 23, 2013 (3 years and 5 months ago)


Genetic engineering and Biotechnology

Topic 4.4

“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
thermal cycling,
consisting of cycles of repeated heating and
cooling of the reaction for
DNA melting and
replication of the DNA.



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

Nobel prize

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

Polymerase is

Nucleotides will bond with exposed bases of the

The original strand has formed a new CDNA.

Cycle is repeated

Gel Electrophoresis

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
molecules through,
however, larger molecules
move more slowly through
it since they cannot
squeeze through the pores
as easily as smaller

Electrophoresis Apparatus

Electrophoresis Technique

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
buffer solution.

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.

Finished Gel

DNA Fingerprinting

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
banding pattern
following a gel

This test is highly
accurate, and the
probability of another
individual possessing
an identical banding
pattern is estimated as

Crime scene

Small amounts of DNA

Amplify the DNA

Two strands are separated

Restriction enzymes

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

DNA Fingerprinting


. Paternity profiling

each band shown on the DNA Profile of a child
must correspond with a band of the father or
the mother.

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
the Romanovs

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

started (3x10

base pairs) in human

In 2003 the sequencing of the human DNA was
99.9% complete.

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
only gene(s)

Provide more info about evolutionary paths by
comparing similarities and differences in genes
between species.

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

Genetic engineering

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
a bacterium

The bacterium produces human protein

Gene transfer

Requires following elements

A vector
to carry the gene into the host

A host cell

Restriction enzymes

DNA ligase

Restriction Enzymes

Restriction enzymes are compounds first
isolated in the 1970's

They function by selectively cutting DNA at
specific sequences

Restriction Enzymes

These cuts usually occur
in the following forms.

The cut can be made
straight across a base
pair sequence resulting in
a "
Blunt End

The cut can be made in
an offset manner leaving
exposed nucleotide
sequences. These
exposed sequences are
called "
Sticky Ends

Blunt End

Sticky end

Gene Splicing

The presence of
sticky ends allows
segments of DNA to
be joined together.

Since DNA strands
which have been cut
by the same
restriction enzyme
can easily bond
together according
to base pairing

Recombinant DNA

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
restriction enzyme.

A plasmid from
E. coli

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
to diabetics.

Two examples
GM crops and

Genetically manipulated organisms

Called transgenic organisms

Flavr Savr

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

Bt corn

G M Maize.

A gene
from Bacillus thuringiensis

Incorporated into maize

Plants produce a toxin that makes them
resistant to insects

Bt crops are grown in US

Bt Corn

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
of insecticide.

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
are lowered.

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.


GM mice

to study the disease polio

Possible treatment and prevention.

Golden Rice

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
yellow color

New kind of rice is now produced

One gene from maize and bacterium

20 times more of beta carotene compared of
Golden Rice.

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
nuclear transfer

Reproductive cloning

Therapeutic cloning

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
and mice.

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
Ian Wilmut

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

The nucleus
egg combination was
stimulated with

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

Reproductive cloning

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

Eg., mare and her cloned foal

Therapeutic cloning

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
specialised tissues.

Other sources of stem cells from umbilical cord
or cells from aborted fetuses.

Aims for cell therapy


Parkinson’s disease

Bone marrow transplants, skin cells for
burn victims

Grow new corneas

Ethical issues of Therapeutic
cloning in humans

Arguments in favor of therapeutic cloning
focus on:

the ability to cure serious diseases with
cell therapy:

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