UNIT 4
Techniques used in
Molecular Biology
Objectives
On completion of this unit students will be able to:
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Outline the steps in Polymerase Chain Reaction
Analyse DNA agarose gel electrophoretograms
Describe DNA hybridization and its application in probe
synthesis
Differentiate between Northern, Southern and Western Blots
Outline the methods of DNA sequencing
Polymerase Chain Reaction
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The PCR technique is basically a primer extension reaction
for amplifying specific nucleic acids in vitro.
PCR will allow a short stretch of DNA (usually fewer than
3000 bp) to be amplified to about a million fold so that one
can determine its size, nucleotide sequence, etc.
The particular stretch of DNA to be amplified is called the
target sequence
The target sequence is identified by a specific pair of DNA
primers (oligonucleotides) usually about 20 nucleotides in
length.
http://stratfeed.cra.wallonie.be/img/page/PCR_web_page5.jpg
Polymerase Chain Reaction
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Primers must be duplicates of nucleotide sequences on
either side of the piece of DNA of interest.
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The exact order of the primers' nucleotides must
already be known.
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Primers can be constructed in the lab, or purchased
from commercial suppliers.
Polymerase Chain Reaction
The cycling reactions :
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There are three major steps in a PCR, which are repeated
for 30 or 40 cycles.
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This is done on an automated cycler, which can heat and
cool the tubes with the reaction mixture in a very short
time.
Thermocycler
Steps in PCR
Three steps in PCR
Polymerase Chain Reaction
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Because both strands are copied during PCR, there is
an exponential increase of the number of copies of the
gene.
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Suppose there is only one copy of the wanted gene
before the cycling starts, after one cycle, there will be 2
copies, after two cycles, there will be 4 copies, three
cycles will result in 8 copies and so on.
Denaturation
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94°C
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During the denaturation, the double strand
melts open to single stranded DNA.
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All enzymatic reactions stop (for example : the
extension from a previous cycle).
Annealing/ Hybridization
54°C :
hydrogen bonds are constantly formed and broken between the
single stranded primer and the single stranded template.
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The more stable bonds last a little bit longer (primers that fit
exactly).
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The polymerase can attach to pieces of double stranded DNA
(template and primer), and starts copying the template.
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Once there are a few bases built in, the hydrogen bond is so
strong between the template and the primer, that it does not break
anymore.
Extension /Elongation
72°C :
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This is the ideal working temperature for the polymerase.
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Primers that are on positions with no exact match, get loose again
(because of the higher temperature) and don't give an extension
of the fragment.
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The bases (complementary to the template) are coupled to the
primer on the 3' side (the polymerase adds dNTP's from 5' to 3').
Polymerase Chain Reaction
The use of a thermostable polymerase allows:
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The dissociation of newly formed complimentary
DNA
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Subsequent annealing or hybridization of primers to
the target sequence with minimal loss of enzymatic
activity.
Polymerase Chain Reaction
Is there a gene copied during PCR and is it the
right size?
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Before the PCR product is used in further applications,
it has to be checked if there is a product formed.
Factors that affect yield:
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quality of the DNA is poor
one of the primers doesn't fit
too much starting template.
Polymerase Chain Reaction
The product is of the right size:
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It is possible that there is a product, for example a band of 500
bases, but the expected gene should be 1800 bases long.
Factors that affect specificity:
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one of the primers probably fits on a part of the gene closer to
the other primer.
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It is also possible that both primers fit on a totally different gene.
Uses of PCR
1.
The method is especially useful for searching out
disease organisms that are difficult or impossible
to culture:
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such as many kinds of bacteria, fungi, and viruses, because it
can generate analyzable quantities of the organism's genetic
material for identification.
Uses of PCR
2. PCR can also be more accurate than standard tests.
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The technique is used to detect bacterial infections by detecting
their DNA
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bacterial ear infection. Sensitive even when culture methods failed to detect
it.
lyme disease, the painful joint inflammation caused by bacteria transmitted
through tick bites., is usually diagnosed on the basis of symptom patterns.
PCR can be used to identify the organism's DNA permitting speedy
treatment that can prevent serious complications.
PCR is the most sensitive and specific test for Helicobacter pylori, the disease
organism now known to cause almost all stomach ulcers.
It can detect the AIDS virus sooner during the first few weeks after
infection than the standard ELISA test. PCR looks directly for the virus‘
unique nucleic acid, instead of the method employed by the standard test,
which looks for indirect evidence that the virus is present by searching for
antibodies the body has made against it..
Uses of PCR
3. The method is also leading to new kinds of genetic
testing.
These tests diagnose not only people with inherited disorders, but
also people who carry deleterious mutations that could be passed
to their children.
Polymerase Chain Reaction
4. PCR can provide enormous peace of mind to people
who are trying to have children
for example, by reassuring anxious parents-to-be that they run
no risk of having a child with a particular genetic disease.
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The technique even saves the lives of babies before they are
born: detect whether the blood groups of mother and fetus are
incompatible. This condition often leads to severe disability and
even death of the fetus, but can be treated successfully in the
womb with enough advance warning-thanks to PCR.
Polymerase Chain Reaction
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Animation PCR
http://www.dnalc.org/ddnalc/resources/pcr.ht
ml
Gel electrophoresis
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a method that separates macromolecules-either nucleic
acids or proteins-on the basis of:
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size
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electric charge
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other physical properties, such as topology.
Pouring a gel
Loading a gel
Loading a gel
http://elchem.kaist.ac.kr/vt/chem-ed/sep/electrop/graphics/eleczone.gif
https://sites.google.com/a/luther.edu/genetics/_/rsrc/1235705828602/st
udents/ashley-dissmore/protocolgel/gel%20box.JPG?height=420&width=325
Gel electrophoresis
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A gel is a colloid in a solid form.
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The term electrophoresis describes the migration of
charged particle under the influence of an electric field.
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Electro refers to the energy of electricity.
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Phoresis, from the Greek verb phoros, means "to carry across."
Gel Electrophoresis
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Thus, gel electrophoresis refers to the technique in
which molecules are forced across a span of gel,
motivated by an electrical current.
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Activated electrodes at either end of the gel provide the
driving force.
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A molecule's properties determine how rapidly an
electric field can move the molecule through a
gelatinous medium.
Gel Electrophoresis
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Many important biological molecules such as amino
acids, peptides, proteins, nucleotides, and nucleic acids,
possess ionisable groups.
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These molecules exist in solution as electrically charged
species either as cations (+) or anions (-) at a given pH.
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The charged particles will migrate either to the cathode
or to the anode depending on the nature of their net
charge.
Gel Electrophoresis
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DNA, is mixed in a buffer solution and applied to a gel.
The electrical current from one electrode repels the molecules
while the other electrode simultaneously attracts the
molecules.
The frictional force of the gel material acts as a "molecular
sieve," separating the molecules by size.
During electrophoresis, macromolecules are forced to move
through the pores when the electrical current is applied.
Gel Electrophoresis
The rate of migration through the electric field
depends on:
 The strength of the field
 The size and shape of the molecules
 The ionic strength and temperature of the buffer in
which the molecules are moving.
Visualization
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After staining, the separated macromolecules in each
lane can be seen as a series of bands spread from one
end of the gel to the other.
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The ladder is a mixture of fragments with known size
to compare with the unknown fragments.
Gel after staining
Gel Electrophoresis
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Animation
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http://207.207.4.198/pub/flash/4/4.html
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`http://www.dnalc.org/ddnalc/resources/electroph
oresis.html
Southern blotting
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Southern blotting was named after Edward M.
Southern who developed this procedure.
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To oversimplify, DNA molecules are transferred
from an agarose onto a membrane.
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Southern blotting is designed to locate a particular
sequence of DNA within a complex mixture.
Southern blotting
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For example, Southern Blotting could be used to
locate a particular gene within an entire genome.
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The amount of DNA needed for this technique is
dependent on the size and specific activity of the
probe. Short probes tend to be more specific.
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Under optimal conditions, you can expect to detect 0.1
pg of the DNA for which you are probing.
Southern blotting
Steps in Southern blotting:
 Digest the DNA with an appropriate restriction enzyme
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Run the digest on an agarose
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Denature the DNA (usually while it is still on the
gel).For example, soak it in about 0.5M NaOH.
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Only ssDNA can transfer.
Southern blotting
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fragments greater than 15 kb are hard to transfer
to the blotting membrane.
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Depurination with HCl (about 0.2M HCl for 15
minutes) takes the purines out, cutting the DNA
into smaller fragments.
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However, that the procedure may also be
hampered by fragments that are too small.
Southern blotting
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Transfer the denatured DNA to the membrane.
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Traditionally, a nitrocellulose membrane is used,
although nylon membrane may be used. Many
scientists feel nylon is better since it binds more
and is less fragile.
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Transfer is usually done by capillary action, which
takes several hours or using a vacuum blot
apparatus which is faster).
Southern blotting
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Capillary action transfer draws the buffer up by
capillary action through the gel into the membrane,
which will bind ssDNA.
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After you transfer your DNA to the membrane, treat it
with UV light. This cross links (via covalent bonds) the
DNA to the membrane.
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(You can also bake nitrocellulose at about 80C for a
couple of hours, but be aware that it is very
combustible.)
Southern blotting
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Probe the membrane with labeled ssDNA. This is also
known as hybridization.
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This process relies on the ssDNA hybridizing
(annealing) to the DNA on the membrane due to the
binding of complementary strands.
Southern blot
Probe Detection
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Visualize your labeled target sequence.
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Probing is often done with 32P labeled ATP,
biotin/streptavidin or a bioluminescent probe.
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If you used a radiolabeled 32P probe, then you would
visualize by autoradiograph.
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Biotin/streptavidin detection is done by colorimetric
methods.
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Bioluminescent visualization uses luminesence.
Radioactive Detection
Probe Detection using Biotin/steptavidin
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streptavidin is added which is an intermediary
compound that will bind to the biotin on the probe.
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Attached to the biotin is an enzyme such as alkaline
phosphatase
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A chromogenic substrate for the enzyme is added eg.
BCIP/NBT (for alkaline phosphatase), which produces
a blue-purple precipitate.
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Therefore, visualization does not require X-ray film or
other specific equipment.
Biotin/Streptavidin detection
http://www.fermentas.com/catalog/kits/img/chromogdet.gif
Biotin/Streptavidin detection
http://www.invitrogen.com/etc/medialib/en/images/ics_organized/brands/mo
lecular-probes.Par.56035.Image.-1.0.1.gif
Northern Blotting
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used for locating a sequence of RNA.
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It is also known as Northern hybridization or RNA
hybridization.
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The procedure for and theory behind Northern blotting is
almost identical to that of Southern blotting, except you
are working with RNA instead of DNA.
Western Blotting
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Western blot analysis can detect one protein in a mixture
of any number of proteins while giving you information
about the size of the protein.
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This method is, however, dependent on the use of a
high-quality antibody directed against a desired protein.
Western Blotting
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So you must be able to produce at least a small portion
of the protein from a cloned DNA fragment to
generate an antibody.
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You will use this antibody as a probe to detect the
protein of interest.
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Western blotting tells you how much protein has
accumulated in cells.
Steps in Western Blotting
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Separate the proteins using SDS-polyacrylamide gel
electrophoresis (also known as SDS-PAGE. This separates
the proteins by size.
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Place a nitrocellulose membrane on the gel and, using
electrophoresis, drive the protein (polypeptide) bands onto
the nitrocellulose membrane.
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You want the negative charge to be on the side of the gel
and the positive charge to be on the side of the
nitrocellulose membrane to drive the negatively charged
proteins over to the positively charged nitrocellulose
membrane.
Steps in Western Blotting
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This gives you a nitrocellulose membrane that is
imprinted with the same protein bands as the gel.
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Incubate the nitrocellulose membrane with a primary
antibody.
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The primary antibody, which is the specific antibody
mentioned above, sticks to your protein and forms an
antibody-protein complex with the protein of interest.
Steps in Western Blotting
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Incubate the nitrocellulose membrane with a secondary
antibody.
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This antibody should be an antibody-enzyme conjugate.
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The secondary antibody should be an antibody against
the primary antibody.
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This means the secondary antibody will "stick" to the
primary antibody, just like the primary antibody "stuck"
to the protein.
Steps in Western Blotting
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The conjugated enzyme is there to allow you to visualize all of
this.
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To actually see your enzyme in action, you'll need to incubate it in
a reaction mix that is specific for your enzyme.
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You will see bands wherever there is a protein-primary antibodysecondary antibody-enzyme complex, or, in other words, wherever
your protein is.
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Put x-ray film on your membrane to detect a flash of light, which
is given off by the enzyme or observe the color change produced
by the enzyme with the substrate.
Western blot procedure
http://www.genscript.com/images/L00204-1.jpg
SDS Gel on left and Western blot on right
http://www.viswagenbiotech.com/images/sds_western_blot_proprep.jpg
Steps in making a primary antibody
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Run the purified protein on an SDS-PAGE gel.
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Stain the gel with KCl.
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The KCl forms a precipitate with the SDS.
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Since the area with the protein has a low concentration
of SDS, the area with the protein will not show a
precipitate.
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This will allow you to see the protein band as a clear
band against a milky white precipitate on the rest of the
gel.
Steps in making a primary antibody
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Carefully cut out the band, crush it and make an emulsion
with 1ml Freund's Complete Adjuvant (which is an oily
substance).
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The complete adjuvant contains bacteria (an immune
stimulant) to increase the immune response.
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Inject this subscapularly into a rabbit.
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This is your first inoculation. Only use the complete
adjuvant for the first inoculation.
Steps in making a primary antibody
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Rest the rabbit for one month, repeat the process using
an incomplete adjuvant. You can expect to see good
antibody titers about 10 days after the second booster.
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Bleed the rabbit. Now you have rabbit antisera.
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To get your primary antibody, dilute the rabbit antisera
in blotto (aka Carnation Nonfat Dry Instant Milk) and
apply it to your nitrocellulose blot. Make sure you dilute
1:500 to 1:100 in blotto.
Steps in making a primary antibody
http://www.blogcdn.com/www.thecancerblog.com/media/2007/01/rat2.jpg
Steps in making secondary antibody
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This is much easier than the procedure for the primary
antibody.
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Grab a catalogue and look for a goat-anti-rabbit
antibody conjugated to horseradish peroxidase (HRP).
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The goat-anti-rabbit is your secondary antibody (the one
that "sticks" to the primary antibody) and the HRP is
the conjugated enzyme that will allow you to visualize
your protein.
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DNA Sequencing
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determination of the precise sequence of
nucleotides in a sample of DNA
first devised in 1975,
has become a powerful technique in molecular
biology
allows analysis of genes at the nucleotide level.
has been applied to many areas of research.
DNA Sequencing
Two methods:
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Enzymatic sequencing/chain termination (or
'Sanger-Coulson-Sequencing')
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The sequence of a single-stranded DNA molecule is
determined by enzymatic synthesis of complementary
polynucleotide chains with these chains terminating at
specific nucleotide positions.
Chemical sequencing method ('Maxam-GilbertSequencing’ Sequencing)
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The sequence of a double-stranded DNA molecule is
determined by treatment with chemicals that cut the
molecule at specific nucleotide positions.
DNA Sequencing
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Both methods were equally popular to begin
with
However the chain termination procedure is
currently preferred, particularly for genome
sequencing.
because the chemicals used in the chemical
degradation method are toxic and therefore
hazardous to the health of the researcher
 because it has been easier to automate chain
termination sequencing.
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Chain termination DNA sequencing
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based on the principle that single-stranded
DNA molecules that differ in length by just a
single nucleotide can be separated from one
another by polyacrylamide gel electrophoresis
The discovery of thermostable DNA
polymerases, which led to the development of
PCR has also resulted in new methodologies for
chain termination sequencing
Chain termination DNA sequencing
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Thermal cycle sequencing has two advantages
over traditional chain termination sequencing:
It uses double-stranded rather than single-stranded
DNA as the starting material.
 Very little template DNA is needed, so the DNA
does not have to be cloned before being sequenced.
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Chain termination DNA sequencing
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Thermal cycle sequencing is carried out in a similar way
to PCR but :
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just one primer is used
each reaction mixture includes one of the ddNTPs
Because there is only one primer, only one of the strands of
the starting molecule is copied
the product accumulates in a linear fashion, not exponentially
The presence of the ddNTP in the reaction mixture causes
chain termination,
the family of resulting strands can be analyzed and the
sequence read by polyacrylamide gel electrophoresis
Chain termination sequencing
The strand synthesis reaction:
 is catalyzed by a DNA polymerase enzyme
 requires the four dNTPs (dATP, dCTP, dGTP ,dTTP)
 would normally continue until several thousand
nucleotides had been polymerized.
 However, this does not occur in a chain termination
sequencing experiment because:
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as well as the four dNTPs, a small amount of a
dideoxynucleotide (e.g. ddATP) is added to the reaction.
The polymerase enzyme does not discriminate between
dNTPs and ddNTPs, so the dideoxynucleotide can be
incorporated into the growing chain, but it then blocks further
elongation because it lacks the 3′-hydroxyl group needed to
form a connection with the next nucleotide.
The result is therefore a set of new chains, all of different
lengths
Chain termination sequencing
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Fluorolabeling has been important in the
development of sequencing methodology,
because the detection system for fluorolabels
has opened the way to automated sequence
reading.
The label is attached to the ddNTPs, with a
different fluorolabel used for each one.
Chain
termination
sequencing
Chain termination sequencing
Chains terminated with A are therefore
labeled with one fluorophore (pink),
 chains terminated with C are labeled with a
second fluorophore (blue),
 chains terminated with G are labeled with
another fluorophore (yellow)
 and chains terminated with T are labeled
with another fluorophore (green).
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Chain termination sequencing
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Therefore, it is possible to :
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carry out the four sequencing reactions - for A, C, G and T - in a single
tube
load all four families of molecules into just one lane of the
polyacrylamide gel, because the fluorescent detector can discriminate
between the different labels and hence determine if each band represents
an A, C, G or T.
The sequence can be read directly as the bands pass in front of
the detector and either printed out in a form readable by eye or
sent straight to a computer for storage.
When combined with robotic devices that prepare the
sequencing reactions and load the gel, the fluorescent detection
system provides a major increase in throughput and avoids errors
that might arise when a sequence is read by eye and then entered
manually into a computer.
Automated DNA Sequencer
Chain
Termination
sequencing
http://users.rcn.com/jkimball.ma.ultranet/B
iologyPages/F/FluorDideoxySeq.gif
Sequence chromatogram
Animation of DNA sequencing
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http://www.dnalc.org/resources/animations/cy
cseq.html
References
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http://users.ugent.be/~avierstr/principles/pcrs
teps.gif
http://www.flmnh.ufl.edu/cowries/PCR.gif