Download Genetic engineering and biotechnology techniques

Types of cloning
 DNA Cloning (Recombinant DNA
Technology)
 Reproductive Cloning (in animals)
 Cloning in agriculture
DNA Cloning review
Bacteria can be
transformed
with recombinant
plasmids;
plasmids (carrying a specific
gene can
then be cloned to
make many copies)
Reproductive Cloning
Two types:
1. Somatic nuclear cell transfer
Animals can be cloned by removing the nucleus from a
somatic cell and injecting it into an egg cell that has had its
nucleus removed (somatic cell nuclear transfer).
After some chemical treatment, the cell starts dividing and
develops into an embryo, is implanted into a surrogate
mother who carries it to term.
Can be fusion of somatic cell and egg cell or microinjection of
only somatic cell nucleus.
Some notes about animal
reproductive cloning
 Dolly the Sheep – this was done to determine if a
specialized somatic cell could be re-programmed to
produce a new organism
 Not a total clone since there is always some
mitochondrial DNA that contributes to the genetic
make-up of an organism
Reproductive cloning
2. Artificial Embryo Twinning
Mimics the natural process of creating identical twins in
a Petri dish.
Embryos are separated into individual cells early on;
those cells develop into embryos which are carried to
terms by surrogate mothers
Therapeutic cloning
 Also called ‘embryo cloning’ – the production of
human embryos for use in research (illegal in Canada)
 Generating stem cells to grow new tissues and organs
Cloning in agriculture
Tissue culture – small piece of
plant grown in test tube, produces
shoots that are identical
OR
Cloning vector – use of a bacterial
plasmid, infects plants (Ti plasmid
can incorporate into host genome)
Foreign genes can be inserted
by using a bacteria,
Agrobacterium tumefaciens,
that can carry a recombinant
plasmid into a plant.
Limitations
 A. tumefaciens only infects dicots (e.g. soybeans,
potatoes, tobacco, etc.)
 Now, DNA molecules can be injected directly into
plant cells (microinjection, electroporation and
particle bombardment)
Applications in agriculture
Designing transgenic (also known as genetically
modified) plants. Why?
 Resist herbicides (round-up ready corn)
 Resist insect pests and diseases (Bt corn)
 Less need for artificial fertilizers
 Resist environmental stresses
 Produce plants with new characteristics (e.g. longer
shelf life)
Examples of transgenic plants
FlavrSavr tomato
Golden rice
Triple stack corn
Applications in agriculture cont’d
 Increasing desirable characteristics in livestock (e.g.
cows, sheep, goats, chickens, etc.). Such as?
 Increased production of meat, milk, eggs and other
animal products
 Better disease resistance
 Changing the fat:lean ratio of meat
Techniques used in
biotechnology research
and applications
RFLP analysis
 Restriction fragment length polymorphism (RFLP –
pronounced rif-lip) is a difference in homologous DNA
sequences
 RFLP analysis is the original method used for profiling
DNA and was used in genetic fingerprinting and
genome mapping before cheaper methods such as PCR
(polymerase chain reaction) and DNA sequencing
came along
How does it work?
DNA samples are digested by restriction enzymes
Then the DNA fragments are run on an electrophoresis
gel
Then an RFLP probe (radioactive DNA probe – sequence
known) is added and will bind to certain matching
bases – this is how they started mapping the human
genome
RFLP Analysis
Comparison of
different lengths
of DNA fragments
produced by
restriction
enzymes to
determine genetic
differences
between
individuals or to
detect disease
A pioneering method but…..
 It takes about one month for the process
 It requires a large amount of DNA
 Still used to detect genetic variation between
individuals, for inherited diseases and more generally
for disease detection
Polymerase Chain Reaction (PCR)
A quick and easy way to make lots of copies of
the DNA we want
Why PCR?
 Often a gene of interest is not in large enough
quantities to analyze
 Much faster than cloning a certain gene or piece of
DNA in bacteria
All happens in a test tube:
1st step: Denaturation
– the break down of double-stranded DNA using heat
2nd step: Annealing
– primers attach to either end; lower temperature
3rd step: Extension
- DNA polymerase extends the strand using nucleotides that
have been added, medium temperature
And the test tubes are in this……
Application
 DNA fingerprinting for forensics and paternity
 Genetic testing for hereditary and infectious diseases
Move aside PCR
 Constant T PCR has been developed
 Recombinase polymerase amplification
 No need for sophisticated PCR equipment
Agarose Electrophoresis of DNA
 used to separate fragments of DNA
 DNA is negatively charged, charge is proportional to
size
 agarose can be used as a molecular strainer (sieve) to
separate the pieces of DNA by size
Gel electrophoresis

A current is run through the
buffer surrounding the gel
and it pushes the DNA away
from the negative anode,
towards the positive cathode
Gel Electrophoresis
A current is run through the buffer surrounding the gel and it pushes
the DNA away from the negative anode, towards the positive cathode
DNA sequencing
 Used to determine the bases along a particular stretch
of DNA
 Was developed during the time of the Human Genome
Project – allowed it to proceed much faster
What do you need?
First we need:
 DNA of interest, with known sequences at the ends
 DNA polymerase
 ATCG primers, modified A, T, C and G in each test
tube.
How does it work?
1st. Double-stranded DNA is denatured
2nd. Primers attach to known ends
3rd. DNA polymerase adds nucleotides
4th. All four nucleotides are mixed into test tubes with
the DNA of interest
continued
 5th. One of the four nucleotides mixtures has an ‘A’
that has been modified – the ‘terminator’.
 Then separate the pieces by running an them on an
agarose gel (electrophoresis) – the place where an A
(adenine) is will show up
 The same happens for the other bases…..