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Biotechnology
Biotechnology
• Biotechnology means using biological
technology to produce useful organisms
such as plants, animals and microorganisms.
• The organisms themselves or the products
which they produce may be useful.
• To do this we use Gene technology to
modify the DNA of these organisms.
• We alter genes, remove genes, add extra
copies of genes or add genes from other
organisms.
The Tools of Biotechnology
• Genes have 2 main components:
– The protein coding region – contains the
nucleotide triplet codes which code for specific
amino acids and the order they are arranged in.
This is a universal code, so in theory the same
protein can be made by any organism.
– The promoter region – controls gene
expression. Regulates in which tissue the gene
should be expressed, at what time and in
response to what stimulus the gene is
transcribed. (only work within Kingdoms.)
Collecting the DNA
• A piece of tissue or a blood sample contain
many cells and can be used to extract
DNA.
• The process of collecting the DNA has 4
steps.
Collecting the DNA
1. Isolate the DNA from the rest of the
cell – cells are mechanically broken open,
then using detergents and enzymes, the
cell walls and membranes (including
nuclear membranes) are broken, thus
releasing the DNA.
Collecting the DNA
2. Remove the unwanted cell debris – done
either by filtering the extract or
centrifuging the mixture. The filtrate
will now contain the DNA along with some
unwanted proteins.
Collecting the DNA
3. Remove the unwanted protein – done by
adding a protease enzyme that breaks
down protein.
(In some research labs phenol will then
be added to destroy all of the unwanted
protein.)
Collecting the DNA
4. Precipitate out the DNA – DNA can now
be precipitated out by pouring a layer of
ice-cool ethanol over the surface of the
filtrate.
The Scissors (Restriction
Enzymes)
• Restriction Endonucleases
• These are enzymes which occur naturally
in bacteria.
• Their function is to protect the bacterial
cells from infection by foreign DNA (viral
DNA) by cutting the DNA into smaller
pieces.
• Restriction enzymes cut DNA at specific
base sequence recognition sites, usually 4
– 8 base pairs in length.
The Scissors (Restriction
Enzymes)
• The base sequence recognition site is a
Palindrome (reads the same in both
directions).
5’ G A A T T C 3’
3’ C T T A A G 5’
• Some restriction enzymes cut DNA
between 2 specific bases at the
recognition site resulting in “blunt ends”
on the two cut ends of the DNA.
Blunt Ends
Restriction Enzymes
• Other restriction enzymes cut DNA in a
staggered manner at the recognition sites,
resulting in “sticky ends” which are
overhangs on the 2 cut ends of the DNA.
Restriction Enzymes
• These Sticky ends are able to bind to
complementary sticky ends on other DNA.
• In this way, segments of DNA can be
inserted into or added to other segments
of DNA, which have been cut by the same
restriction enzymes to produce matching
sticky ends.
Restriction Enzymes
• Restriction enzymes are named from the
genus, species and strain of the bacterium
from which they were isolated.
• E.g. Eco R1 is from E. coli Strain RY13 and
1 = the first restriction enzyme isolated
from E.coli.
• There are now more than 400 restriction
enzymes in use.
To Do
• Biotech book A pg 10 – 14 Activities 1-3
The Glue – Ligase Enzymes
• Combining the sticky ends of 2 different
DNA strands is only temporary, because
only a few H-bonds hold the ends
together.
• The joins can be made permanent by the
use of DNA Ligase Enzymes.
• These catalyse the formation of
phosphodiester bonds between the
phosphates and the sugars on the sides of
the DNA ladder during replication and
repair.
Ligase
• http://www.slic2.wsu.edu:82/hurlbert/mic
ro101/images/LigaseAnimation6.gif
Cloning
• A clone is a vector molecule carrying a
unique fragment of DNA.
• It can also be used to describe a colony of
cells derived from a single cell, which has
in turn received a single plasmid molecule.
• (plasmids – small circular double stranded
pieces of DNA found mostly in bacteria.)
DNA Carriers
• A Vector is a vehicle that carries DNA
into a host cell.
• The engineered piece of DNA has to be
put back into the cell in order to function,
as it needs the rest of the cell machinery
to function.
• Can be done by natural vectors (bacteria,
viruses, yeast) or when natural vectors
can’t be used (in plant and animal cells)
electroporation or a DNA gun can be used.
Bacteria as Vectors
• The usual vectors used are the plasmids
from bacteria.
• These are circles of DNA that can be
removed from the bacteria and are
opened using restriction enzymes to leave
sticky ends.
• The piece of DNA of interest can be cut
with the same restriction enzymes, and
inserted into the plasmid.
• This can be placed back in the bacteria.
Bacteria as Vectors
• The bacteria divides, replicating the
foreign DNA until you have many copies.
• These can now be used in different ways.
– To produce many copies of the gene of interest,
which could then be isolated and placed into
another organism such as a plant.
– To produce the protein that the gene of interest
codes for, e.g. insulin and growth hormone.
– The engineered bacteria may have a new
function, such as cleaning up oil spills.
Viruses as Vectors
• A virus is a string of DNA (RNA in some
viruses), that is covered with a protein
coat.
• Virus that attack bacteria are called
Bacteriophages or phage for short.
• New DNA can be spliced into the virus
DNA, which is then returned to the virus
coat.
Viruses as Vectors
• The Phage can then infect a bacteria.
• Inside the bacteria the DNA replicates,
using the bacterial cell machinery, to make
new phage particles.
• These in turn infect more bacteria; and so
on.
• It is possible to carry DNA into animal
cells using viruses.
Viruses as Vectors
• Viruses can be used to clone larger
fragments of DNA then plasmids.
• They are also often host specific, will only
invade certain cells.
• E.g. a respiratory virus that invades the
lungs has been used experimentally to
insert a healthy cystic fibrosis gene into
the lungs of rats and the nassal passages
of humans.
Yeast as a Vector
• Sometimes the protein to be made is too
complicated for a prokaryote, so a
eukaryote cell needs to be used.
• Yeast has plasmids so it is ideal.
Electroporation
• An electric current is used to force DNA
across a cell membrane.
• This is useful for introducing plasmids into
eukaryotic cells.
DNA Gun
• The DNA of interest is coated onto
microscopic pellets (gold or tungsten) and
fired into a cell with a gun.
• This is useful for introducing DNA into
plant cells that are not susceptible to
infection.
DNA Synthesis
• We are now able to synthesise small, onesided strands of DNA, about 100 bases
long, called Oligonucleotides.
• Scientists simply type in the sequence
they want into a computer joined to the
synthesiser, and the machine joins
nucleotides together in the right order.
DNA Synthesis
• Chemically-made small oligonucleotides are
usually called Synthetic Oligonucleotides.
• They are important for making a primer
for PCR.
• They can also be used as Gene Probes: if
you know the sequence of nucleotides of
the gene you want, you can make a
complimentary oligonucleotide that will
attach to a DNA if there is a perfect
match in their sequences.
DNA Synthesis
• Oligonucleotides are also useful in finding
a mutation within a gene where as little as
1 base has changed.
• This is important in genetic screening,
because a single base change can cause an
abnormal protein to be made which can
lead to a genetic disease.
Polymerase Chain Reaction
• This is a method by which a minute piece
of DNA can be quickly copied many times
in vitro.
• The advantage that this method has over
cloning is that billions of copies of a piece
of DNA can be made in hours as opposed
to weeks.
Starting Materials
• Pieces of double stranded DNA containing
the nucleotide sequence to be copied.
• DNA polymerase enzymes
• A good supply of all 4 nucleotides.
• Primers (synthetic oligonucleotides with
sequences that are complementary to the
ends of the required piece of DNA).
The Method for PCR
• The DNA is heated briefly to allow it to
unwind and separate.
• Then it is cooled to allow primers to bind
to the ends of the desired sequence with
hydrogen bonds – one primer on each
strand of DNA at the 3’ end
• DNA polymerase adds new nucleotides
from the primer onwards, using the DNA
as a template (DNA synthesis).
The Method for PCR
• The DNA has now been doubled.
• The process is repeated over and over,
doubling the amount of the target DNA
each time.
• Each cycle takes about 5 mins.