Biotechnology:

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23 Οκτ 2013 (πριν από 3 χρόνια και 10 μήνες)

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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
enzymatic
replication of the DNA.


PCR


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.


KARY MULLIS
-
developed in 1983


Nobel prize
-
1993




http://learn.genetics.utah.edu/content/labs/pcr/


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
added


Nucleotides will bond with exposed bases of the
ssDNA


The original strand has formed a new CDNA.


Cycle is repeated



Gel Electrophoresis


Gel electrophoresis is a
technique used to
separate fragments of
DNA.



Separates fragments as a
function of size and
charge.



Most types use
Agarose

to separate fragments.



Agarose

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
molecules.

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


http://www.dnalc.org/resources/animations
/gelelectrophoresis.html


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
individuals.

Structural genes are often separated by large regions of repeating base
pairs.

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
electrophoresis.



This test is highly
accurate, and the
probability of another
individual possessing
an identical banding
pattern is estimated as
around
1:14,000,000,000.

Crime scene


Small amounts of DNA


Amplify the DNA
-
PCR


Two strands are separated


Restriction enzymes
-

endonucleases used to
cut


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

Application

a
. 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
people.


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


Conclusion
-

Bodies were likely to be the Romanov family


see



http://en.wikipedia.org/wiki/Genetic_
fingerprinting
------
for some ideas of
problems when using DNA profiling as
evidence.

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


1990
-
started (3x10
9

base pairs) in human
DNA


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
disease


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
insulin.






Gene transfer


Requires following elements


A vector
-
Plasmid
-
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
rules.

Recombinant DNA

Gene Splicing contd..


This allows for genes to be
"cut & pasted" between
organisms. This can be seen
with production of human
insulin.


The DNA sequence of insulin
is identified and cut out using a
restriction enzyme.


A plasmid from
E. coli

is
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
animals


GMO
-
Genetically manipulated organisms


Called transgenic organisms

Flavr Savr


1994
-
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
(Bt)


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
reduced.



Bt corn is slightly more expensive, but the
difference is less than one extra application
of insecticide.


Non
-
Bt corn needs to be checked often for
signs of ECB
-

less checking needed for Bt
corn.


Less insecticide needed means less impact on
the environment and lower health risks for the
worker(s)

•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.


examples


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
bacterium


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
-
globalisationists.

Cloning

clone


Group of genetically identical organisms or
group of cells derived from a single cell


Using differentiated cells is mostly somatic cells
nuclear transfer
-
SCNT.



Reproductive cloning



Therapeutic cloning



Cloning: What it is



Cloning

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
Dolly
.


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


Dolly




In 1997, cloning was revolutionized
when
Ian Wilmut

and his colleagues at
the Roslin Institute in Edinburgh,
Scotland, successfully cloned a sheep
named
Dolly
. Dolly was the first cloned
mammal.


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
ewe.


The nucleus
-
egg combination was
stimulated with
electricity

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


http://learn.genetics.utah.edu/content/tech/
cloning/

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 successfull.eg


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


Used

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
diabetes.

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
anyway)



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
its.