SNC4M
Biotechnology Unit
Cloning and biotechnology technique
Types of cloning
1. DNA cloning (using recombinant DNA Technology)
Bacteria can be ________________________ with recombinant plasmids; plasmids (carrying a
specific gene can then be cloned (simply grown in large quantities) to make many copies). Remember
recombinant plasmids (the recombination of the DNA of different organisms) are made using
restriction enzymes.
2. Reproductive cloning
Two types:
1. Somatic nuclear cell transfer
Animals can be _______________________________by removing the nucleus from a somatic cell
and injecting it
into an egg cell that has had its nucleus ____________________________________ (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.
This type of cloning can refer to the _____________ of a somatic cell and egg cell or the
______________________________________ of the somatic cell nucleus only (done manually
under a microscope).
SNC4M
Biotechnology Unit
Some notes about animal reproductive cloning

Dolly the Sheep – this was done to determine if a specialized somatic cell could be
__________________________________________ to produce a new organism

Not a total clone since there is always some _________________________________
DNA that contributes to the genetic make-up of an organism
2. Artificial Embryo Twinning
This process mimics the natural process of creating _______________________ twins in a Petri
dish.
Embryos are ________________________________- into individual cells early on; those cells
develop into embryos which are carried to terms by surrogate mothers. Not a good survival rate of
embryos due to chemical and electrical treatment.
3. Therapeutic cloning

Also called ‘embryo cloning’ – the production of _________________________________
for use in research (illegal in Canada)

This also refers to generating _________________________ to grow new tissues and
organs
3. Cloning in agriculture - plants
1. Tissue culture – small pieces of plant grown in test tube, produces shoots that are identical
OR
2. Transformation of plant cell – through a plasmid that carrier the foreign DNA or other
techniques such as __________________________________. This is the common way to produce
_______________________________________ plants (plants with foreign DNA).
SNC4M
Biotechnology Unit
Foreign genes can be ________________________by using a bacteria, Agrobacterium
tumefaciens, that can carry a recombinant plasmid into a plant.
Limitations
A. tumefaciens (a natural parasite of some plants), only infects dicots (e.g. soybeans, potatoes,
tobacco, etc.).
Now, DNA molecules can be injected directly into plant cells via _____________________,
electroporation and particle bombardment
Particle bombardment (the ________________________ method) using a gene gun – DNA is
bound to tiny particles of gold or tungsten, which are ‘shot’ into the cell.
Applications of genetic engineering in agriculture
Designing transgenic (also known as genetically modified) plants. Why?

Resist _________________________________ (round-up ready corn)

Resist insect pests and diseases (Bt corn)

Less need for artificial fertilizers

Resist ___________________________________--- stresses

Produce plants with new characteristics (e.g. longer shelf life)
SNC4M
Biotechnology Unit
Examples:
Golden rice – the gene for the precursor to vitamin A was added (to counteract a deficiency in some
parts of the world)
FlavrSavr tomato – a gene was added that interferes with the production of an enzyme that
degrades pectin in the cell wall, therefore prolonging shelf life
Bt corn – a toxin produced by the bacteria, Bacillus thurigiens (Bt) was introduced into corn in order
to resist attack from the European corn borer larvae.
Other application in agriculture cont’d
Genetic engineering is used to increase the ________________________ 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
SNC4M
Biotechnology Unit
Techniques used in biotechnology
1. RFLP analysis

Restriction fragment length polymorphism analysis (RFLP – pronounced rif-lip) is a technique
based on ________________________ in homologous DNA sequences

RFLP analysis is the original method used for profiling DNA and was used in
________________________________ and genome mapping before cheaper methods
such as PCR (polymerase chain reaction) and DNA sequencing came along Still used to detect
genetic variation between individuals – e.g. for inherited diseases and sometimes for disease
detection
How does it work?
First, DNA samples are digested by restriction enzymes, then the DNA fragments are run on an
electrophoresis gel. Subsequently, 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
A pioneering method but:

It takes about one month for the process

It requires a _______________ amount of DNA

Still used to detect genetic variation between individuals, for inherited diseases and more
generally for disease detection
2. Polymerase Chain Reaction – the fast way to make lots of copies (amplification) of a DNA
fragment/gene of interest

Often a gene of interest is not in large enough quantities to analyze

Much __________________________ than ____________________ a certain gene or
piece of DNA in bacteria
All happens in a test tube:
1st step: _________________________ – the break down of double-stranded DNA using heat
2nd step: Annealing – primers attach to either end; lower temperature
3rd step: Extension - DNA polymerase _____________________ the strand using nucleotides
that have been added, medium temperature
SNC4M
Biotechnology Unit
Used whenever larger quantities of DNA are required – could be for genetic testing, DNA
fingerprinting for forensics and paternity, etc.
Move aside PCR

Constant T PCR has been developed

Recombinase polymerase amplification

No need for sophisticated PCR equipment
3. Agarose Gel electrophoresis



used to ________________ 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
__________
SNC4M
Biotechnology Unit
4. 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 faster
What is needed?

the DNA of interest, with ___________________ sequences at the end

DNA polymerase and ATCG primers

_______________________ A, T, C and G in each test tube
How does it work?
1st. - the double-stranded DNA is ____________________________
2nd. the primers attach to known ends
3rd DNA polymerase is added – it adds ________________________________-4th. All four nucleotides are mixed into test tubes with the DNA of interest
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…..