Download Biotechnology toolkit part 2

Biotechnology toolkit part 2
5. Gel Electrophoresis
Electrophoresis means to carry with electricity. It uses the fact that the
phosphate groups in DNA are negatively charged.
In an electric field DNA fragments migrate towards the positive electrode
(anode). By placing the fragments in a gel, fragments encounter resistance
that depends on their size. The smaller fragments have less resistance and
move faster through the gel. Larger fragments encounter more resistance and
move slower. A sample of digested DNA can be separated into discrete bands
containing millions of strands of DNA of identical length. A dye is added to the
DNA sample, which moves slightly faster than the shortest fragments so you
can see how far the bands have travelled. The DNA can be visualised by
adding ethidium bromide, which fluoresces under UV light.
Gel electrophoresis can be used to measure the lengths of fragments of DNA.
Marker bands with known sizes of fragments are run on the gel at the same
time as the unknown DNA fragments.
Electrophoresis
Here we can separate a mixture of molecules using an electric current.
Fragments of DNA separated to form a "fingerprint" is one application of this
technique.
(Featured in the New Zealand Science Teacher journal, No 103, 2003. ISSN
0110-7801) Found at
http://www.nexusresearchgroup.com/fun_science/electrophoresis.htm
Materials: Soap dish; 9 volt batteries; Aluminium foil OR nichrome wire;
Agarose OR Agar; Baking Soda; Ice-cream container lid; Glycerol; Droppers
or Pasteur pipettes; Test mixture (could be plant indicator material that is
concentrated by evaporation or Human cheek cell DNA); Methylene Blue
(only if DNA was your test mixture)
BUFFER: Dissolve about 1/4 teaspoon Baking Soda in 1 1/4 cups water (3 g
in 300ml water). In the lab you can make a 1% solution by dissolving 1g in
100ml water.
GEL: Agarose is traditionally used but may be expensive. Make a 1% agarose
gel by putting about 1/4 teaspoon of agarose in 1/4-cup buffer (1g in 100ml).
Alternatively use agar (not nutrient agar) in its place. I have often used a 0.8%
agar gel (0.8g in 100ml).
GEL COMB: Use a thin plastic lid similar to an ice-cream tub lid to cut a
"comb" that will rest on the sides of the dish but have rectangular "teeth" with
square ends pointing downward. The 5mm wide teeth need to be cut to a
length that will stop 2 or 3mm from the bottom of the soap dish. See if you can
get four or more teeth on a comb.
ELECTROPHORESIS CHAMBER: Use a soap dish lid or its base.
1
Microwave the agarose or agar gel solution on High until all has
dissolved (this may take about 5 minutes). Allow to cool to about 50ºC (able to
be held in hand with out burning). If it gets too cool and starts to set, simply
reheat! You can do this as many times as required.
2
Pour your "hand hot" gel solution into your dish. You need a gel
between 5 and 7mm thick, no more. Immediately place you comb into the gel
about 2 cm from one end. This will form "wells" in which to place your sample
mixtures.
3
Leave to set on a level surface. Carefully remove the comb. We
now want to prevent the gel from being distorted by the bubbles that form at
the electrodes. Use a blade to cut out a 1cm wide strip of gel from opposite
ends of the dish where the electrodes will go. Don't damage the wells that
should now be 1cm from the new edge.
4
At each end, place a wide strip of aluminium foil covering the
interior sides to act as electrodes, or alternatively, place a length of nichrome
wire that overlaps one edge of the dish for attachment of an alligator clip.
5
HINT: To speed things up you could pour several gels and store
in the fridge with plastic wrap covering the top. They will keep for quite a few
days.
LOADING THE WELLS:
1
Flood the gel with buffer (ice cold sometimes helps) so that the
gel is covered to a depth of about 3mm.
2
Mix 2 drops of glycerol with 6 drops of your sample on a slide.
The glycerol will make your sample more dense so it will fall into the well
rather than diffuse into the buffer and be "lost". Colourless samples can have
a drop of Bromphenol Blue added.
3
Collect some of your sample in a dropper or Pasteur pipette and
place the tip just under the surface of the buffer above a well. Slowly fill the
well. Coloured samples of plant material are easy to monitor.
4
Fill the other wells in a similar manner with the rest of your test
samples. Don't forget to keep a record of what sample was in which well!
RUNNING THE GEL:
1
Use an alligator clip to connect the electrode closest to the wells
to the negative terminal of one of the 9-volt batteries. Connect 4 other
batteries in series by stacking and connecting oppositely charged terminals in
a pyramid fashion. The remaining positive terminal is connected to the other
electrode with another alligator clip.
2
After a while you should see bubbles forming at the electrodes.
Leave the gel until your samples have separated into distinct bands. If you
had to add Bromophenol Blue wait till it has almost run to the end of the gel.
3
If you have used plant or animal DNA in your sample, stain the
gel with Methylene Blue (0.02 g in 100ml water). Stain for one hour (at least)
then rinse in water.
6. DNA Probes
Every gene contains a unique sequence of the four bases: adenine (A),
cytosine (C), guanine (G) and thymine (T). We can test to see if a specific
gene is present in a person’s genetic make-up by searching for its unique
base sequence.
The search uses a gene probe, which is a piece of single-stranded DNA. The
design of a probe uses the fact that when DNA strands pair up, adenine only
pairs with thymine and cytosine only pairs with guanine. The base sequence
on the probe matches the unique sequence in the gene that the probe is
designed for.
To test a DNA sample using a gene probe, the DNA is first treated so that
each of the double-stranded DNA molecules unzips into single strands. The
probe is then added to the solution. Because of the way the bases pair up, the
probe will attach itself only to the section of DNA that contains a base
sequence that matches the probe’s sequence.
Probes are constructed with a radioactive or a fluorescent section, or tag, in
them, so that they can be detected after attaching to the DNA. Detecting the
probe gives us information about which chromosome the gene is on, and
where the gene is on that chromosome.
We know the base sequences in a number of disease-causing genes, and
can find out if they are present using probes specifically designed for them.
http://www.industry.gov.au/biotechnologyonline/biotec/findgene.cfm
7. PCR (Polymerase Chain Reaction)
This is one of the most useful techniques in the genetic engineering toolkit.
It is a method in which a piece of DNA is copied many times in vitro. The DNA
is can be increased by a billion-fold or more. To study genes, researchers
need large amounts of DNA to work with. PCR copies the cell’s natural ability
to replicate its DNA and can generate billions of copies within a couple of
hours.
There are four main stages:
1.
The DNA to be copied is heated, which causes the paired strands to
separate. The resulting single strands are now accessible to primers (short
lengths of DNA). This is called denaturing.
2.
Large amounts of primers are added to the single strands of DNA. The
primers bind to complementary matching sequences along the DNA
sequence, in front of the gene that is to be copied. The reaction mixture is
then cooled which allows double-stranded DNA to form again. Because of the
large amounts of primers, the two strands will always bind to primers, instead
of to each other. This is called annealing.
3.
DNA polymerase is added to the mixture. This is an enzyme that
makes DNA strands. It can synthesize strands from all the DNA primer
combinations and dramatically increases the amount of DNA present. This is
called extending.
One enzyme used in PCR is called Taq polymerase which originally came
from a bacterium (Thermus aquaticus) that lives in hot springs. It can
withstand the high temperature necessary for DNA strand separation and
therefore, can be left in the reaction and still functions.
4.
The above steps are repeated until enough DNA is obtained. In
subsequent cycles, the process of denaturing, annealing and extending are
repeated to make additional DNA copies. After three cycles, the target
sequence defined by the primers begins to accumulate. After 30 cycles, as
many as a billion copies of the target sequence are produced from a single
starting molecule.
This whole process is automated and happens very quickly. The reaction
occurs in a small tube which is placed inside a specialised machine which can
make the big temperature adjustments quickly.
The materials needed for PCR are
 Pieces of double stranded DNA containing the nucleotide sequence
that is to be amplified (copied)- can be a gene or a non-coding
sequence, to act as a template.
 DNA polymerase enzymes (from thermophilic organisms)
 A supply of all 4 nucleotides (for making the new DNA)
 Two oligonucleotide primers (complementary to the ends of the
target DNA you want to amplify)
Uses of PCR
1. Used by forensic scientists with a very small piece of tissue- to identify
criminals and eliminate the innocent.
2. Used by anthropologists and archaeologists to check ancient fossils.
3. Used by scientists to identify is a person has that particular gene. Eg
Cystic fibrosis
4. Can be used to identify viral genes much more quickly than other
methods (eg HIV)
5. Can be used to rapidly identify any prenatal genetic disorder from a few
foetal cells
6. Can detect cancer cells from a few cells. (Eg bowel cancer)
7. Can identify an unknown body or skeleton by amplifying the DNA to be
checked against other people to see if there is a match.
http://www.dnai.org/b/index.html
8. DNA Sequencing
DNA sequencing is the determination of a base sequence of a length of NDA.
The method for working out the sequence was discovered by a British
Scientist Frederick Sanger.
It involves making a new strand of DNA complimentary to the strand that is
unknown, and randomly incorporating a modified nucleotide called
dideoxyribose nucleotide (i.e. minus two oxygens). When this is incorporated
into a DNA strand, it stops the synthesis at that point.
This technique requires
* DNA polymerase
* Unknown DNA
* Radioactive primer
*The four normal nucleotides, A, T, C and G (nucleotide triphosphates)
* Nucleotide analogues, “fake” nucleotides, which lack an OH group on
carbon 3. These can be added to the 3’ end of a pre-existing chain but cannot
accept any more since their carbon #3 has no OH group.
Suppose the analogue G* is included with the normal ingredients. DNA
replication occurs along the thousands of copies of the DNA until it is halted
by incorporation of the analogue. Since the concentration of the G* is much
lower than that of the normal nucleotide, it is random chance that the G* is
incorporated and not the normal G. The result is a mixed population of
partially replicated DNA fragments (of different sizes), all ending in a
nucleotide analogue.
Uses of DNA sequencing
*Uses in laboratories to map genomes of organisms, including the human
genome.
*Having found a gene, the gene can be analysed and a probe made to fit a
small part of it. This probe can then search the DNA for a potential carrier of
the disease, hybridise to it and show up by radioactive displays on an x-ray
film.
*It can be used to analyse the amino acid sequence in a protein.
*The gene sequence tells scientist where restriction enzyme sites are located;
the right restriction enzyme can be chosen to cut a gene if necessary
* The results of DNA sequencing can be stored in a computer data bank, very
important to help understand the genes and also position of shorter pieces of
DNA that control the sequences. (BLAST)
*Sequencing can be used to compare DNA from other samples from similar
animal or organisms. This can led to new and often more accurate, tool to
determine evolutionary relationships between organisms.
*Can tells scientists if the DNA they have used has mutated or been
incorporated in the correct place.
9. Gene Cloning
One of the most important requirements in gene technology is to be able to
replicate a desired sequence. Although PCR is an immensely powerful tool,
the most convenient method uses the metabolism of a host organism- usually
a bacterium.
To get the DNA of interest into a host bacterium it must first be inserted into a
larger piece of DNA. This is then replicated into the bacterium, alongside the
gene on interest. After many reproductive cycles large numbers of identical
copies of the gene are produced. A series of identical copies is called a
clone. The bacterial or viral DNA acts as a carrier and is called a cloning
vector.
Sources of the genes for cloning.
1. DNA collected directly from an organism. Scientists split up DNA and
make a recombinant DNA in bacteria plasmids. This is a somewhat
“shotgun” method and you end up with thousands of plasmids, each
carrying copies of certain fragments of the foreign DNA. This is called a
genomic library,
2. Complementary DNA. Not every piece of DNA within a gene actually
codes for a protein. Those not coding for anything are called introns.
The pieces that code for the protein are called exons. All that is left on
the mRNA strand once it is in the cytoplasm is the exons. Scientists
need to make a DNA strand that has the introns removed because
bacteria and prokaryotic cells cannot read and edit out the introns.
10. Plasmids
The term vector is used to describe any vehicle that carries DNA into a host
cell.
The most usual vectors are the plasmids from bacteria. Plasmids are circular
pieces of DNA that are small (around 5000bp). Plasmids can be removed
from the bacteria and opened by restriction enzymes to leave sticky ends. The
piece of DNA of interest can be cut with the same restriction enzyme and
inserted into the plasmid. This can then be put back into the bacteria.
The bacterium divides; every time it does it replicates the foreign DNA until
you have many copies.
Plasmids are useful because
 They can be taken up by bacteria of the same or even another
species. The update of DNA by bacteria is the basis for
transformation and is the mechanism by which resistance to
antibiotics can spread from one species of bacterium to another.
 Because they carry genes for resistance to antibiotics, they can be
used as markers. A gene of interest is incorporated into a plasmid
carrying a gene for antibiotic resistance (or ß-gal) and the plasmid
transported onto a medium containing that antibiotic. Any survivors
must have received the plasmid (to be able to survive on the
medium) and hence the passenger gene.
Process
1. DNA of interest is extracted and amplified. The DNA is then digested
with the same restriction enzyme as will be used to digest the plasmid.
2. The plasmid is extracted from a bacterium and digested with a
restriction enzyme.
3. The plasmid and DNA of interest are added together along with a
ligase.
4. The plasmids are transformed into bacteria. Plasmids are added to a
suspension of bacteria. Transformation (the uptake of plasmids) is
promoted by a chilling followed by a brief warming to 40ºC, a small
proportion of the bacteria are transformed by taking up the DNA.
5. The bacteria and plasmid solution is grown on agar plates. The gar will
have an antibiotic to select those bacteria, which have taken up the
plasmid and may also contain lactose to indicate those transformed
bacteria as well.
6. The bacteria that indicate that they have taken up the plasmid are
checked by restriction enzyme digests and DNA sequencing. The
correct plasmids are then maintained in cells as self-replicating units.
11. Bacteriophages
Bacteriophages are viruses that attack bacteria. Bacteriophages invade a host
bacterium and cause the lysis of the lost cell and release hundreds of new
phage particles. Each of these can then infect neighbouring cells, and so on.
Each cycle takes about 20 minutes. When this happens in a dense population
on an agar plate, a clear area or plaque is produced on the agar. A plaque
marks the position of millions of phage particles.
The most commonly used phage is phage .
Process
1. The phage particles are firstly broken up into their protein and DNA
components.
2. A non-essential part of the phage DNA is removed to make room for
the passenger DNA.
3. The DNA of interest and the phage DNA are digested with the same
restriction enzyme.
4. The viral and DNA of interest are mixed and after the ends have been
annealed they are covalently joined using DNA ligase.
5. The protein coat is then added back and the phage is then used to
infect the cells of the recipient bacterial.
12. DNA Profiling
This is a technique that makes it possible to determine relationships between
individuals by comparing their DNA
It can be used
 to determine paternity
 in rape cases to determine guilty or innocent
 has been used on Europe to determine whether they have relatives
in the country he or she is trying to enter.
 Is important in captive breeding to remove chances of inbreeding.
DNA profiling is better than using blood group test because
 Blood groups aren’t specific and can only be used to eliminate a
subject
 Any tissue contains cells – skin, hair root, bone etc, which can
serve as a DNA source.
Exons are the coding parts of a gene and introns are the non-coding parts of
a gene which are removed before translation (splicing). About 90% of the
human genome has no known function and mainly consists on intron. Exons
that code for the amino acid sequence in essential proteins vary little, since
changes in that gene is likely to be harmful. Introns sequences however vary
greatly in a population and form the basis for DNA profiling.
Much of the intron DNA contains regions called minisatellites.
A minisatellite can consist of short sequences of 20-100 nucleotides that
repeat. These are called Variable Number Tandem Repeats regions (VNTRs).
It may also contain repeats of only 2-4 bases, showing a variation on only 515 repeats. These are called Short Tandem Repeats (STRs)
These form the basis for DNA profiling. VNTRs and STRs are inherited in a
Mendelian way. Each person inherits one VNTR from each parent. VNTRs
and STRs give scientists a tool to compare DNA from two or more related
organisms to see how they are related. Each person has a unique genetic
fingerprint (excluding identical twins). The probability of 2 people having the
same genetic fingerprint (excluding identical twins) is 1:30,000 million
In practise the whole genome is not analyses, but only about 5 to 10 tiny
regions of the genome. The regions chosen are known to be highly variable
from one person to another, so the chances that two people would be the
same is extremely small.
Making a Genetic Profile
1. A sample of the DNA of the individual is digested with a restriction
enzyme, which does not attack any site within a repeated sequence, so
the repeats are intact.
2. The fragments are then separated by gel electrophoresis. At this stage
they are invisible and are also greatly outnumbered by other DNA
fragments.
3. The gel is then subjected to Southern Blotting so the DNA is
transferred to a membrane.
4. A DNA probe is used to identify the repeat fragments. The probe binds
to the repeat fragments, labelling them.
5. The non-bound probe is washed away and an x-ray film laid over the
filter.
6. After developing the film, the positions of the repeat sequences are
revealed as dark bands on the film. The spacing between the
fragments indicates their rates of movement through the gel, and
hence their relative sizes.
13. Transgenesis
Transgenesis involves the removal of DNA from one organism and splicing it
into the genome of a different species.
Although the genetic code is the same in bacteria, fungi, plants and animals,
there are a number of important differences in the way proteins are made,
which complicate the effective transfer of genes between eukaryotes and
prokaryotes, there are also other technical limitations.
Method of Transgenesis
Transgenesis involves 5 stages:
1. Obtaining DNA
There are 2 ways of isolating a gene
*Indirectly by making the DNA for the gene from its mRNA
*Directly from the DNA
2. Inserting Fragments into Cloning Vector
The recombinant DNA so formed is inserted into a host organism within which
the recombinant DNA is replicated. (Bacteriophage = infects cells, Plasmid =
bacterial transformation). The result is a large number of copies of the
recombinant DNA in either the bacterial colonies or plaques.
3. The Clone Containing the Desired Gene is Isolated
Having obtained a cDNA or genomic library, the correct clone must be
identified. The most common way is to use a gene probe.
4. Introducing Genes into Animal Cells
Introducing foreign DNA into mammalian cells is transfection. The most
common methods are
1. Microinjection: DNA is injected into nucleus of recipient cell.
2. Electroploration: Cells are given electric pulse, which causes minute
holes to form in plasma membrane so DNA can be taken up from the
medium.
3. Virus: Use a virus genetically engineered to become harmless as a
carrier.
4. Lipofection: DNA is coated in liposome’s, which are taken up by the
plasma membrane.
4. Introducing Genes into Plant Cells
Plants have a great advantage over animal cells in that their cells are
totipotent, meaning that any cell can grow into an entire plant if given the
appropriate chemical treatment.
5. Getting the Cells into the Animal or Human
If a gene is inserted into the nucleus of a fertilised egg before it starts to
divide, then every cell from then on will carry the new gene.
Genes can be transferred into either a fertilised egg or a body cell. Ethical
considerations have prevented experiments with fertilised eggs in humans.
Genes are introduced into the cells by 2 methods:
In vitro
This is done outside the body. Cells are removed from a patient and then
infected with a virus carrying the new gene, or a DNA gun can fire the DNA
into the cells. The resulting cell is then put back into the body.
In vivo
This is done inside the body. A retrovirus is used to carry the new gene
(limited to 7000bp) and is inserted into the body.
Examples
Placing a gene for 1-antitrypsin with control genes so that it is only
expressed in the mammary glands of a sheep, goat or cow.
Injecting a cloned growth hormone gene into the nuclei of rainbow trout or
salmon, so that it makes them grow in 1 year to a size that normally takes 23years.
Transgenic animals such as mice can be engineered to carry human diseases
so that treatments can be developed to use them.
Used to treat severe combined immunodeficiency disease that results from an
adenosine deaminase deficiency (ADA). (Boy in the bubble)
Cystic Fibrosis genes have been incorporated into lung cells using an
adenovirus but it doesn’t incorporate very well into the lung cells so the
treatment needs to be repeated.
5. Growing Transgenic Plants in Tissue Culture
A tiny portion of plant called an explant usually buds, discs of leaf, stem or
root tissue. Genes are introduced into the explants using Agrobacterium.
Producing plants form tissue culture has important advantages:
Large numbers can be produced very quickly
All are identical to the genotype of the ‘parent’
It can be done at any time of the year
Can select for plants that are resistant to certain environmental stress, eg cold
=> resistant to frost
Examples
Herbicide resistance- can spray crop with weed-killer but it wont harm the
crop
Insect resistance- Toxic protein (BT) inhibits the insects gut enzymes so they
die
Virus resistance- uses the pathogens genes to block replications
Long-life fruit- eg Flavr Savr tomatoes, has antisense strand that prevents fruit
from ripening