Fluorescent Western Blot Kit: MaxTagâ„¢ for IRDYE Western Blot
TECHNIQUES USED IN GENETIC...
Transcript of TECHNIQUES USED IN GENETIC...
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TECHNIQUES USED IN GENETIC ENGINEERING 1
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ELECTROFORESIS
BLOTTING
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Uses of DNA Profiling
• DNA profiling is used to solve crimes and medical problems
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Crime
• The DNA profile of each individual is highly specific.
• The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins).
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Biological materials used for DNA profiling
• Blood • Hair • Saliva • Semen • Body tissue cells • DNA samples have been
obtained from vaginal cells transferred to the outside of a condom during sexual intercourse.
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DNA Profiling can solve crimes
• The pattern of the DNA profile is then compared with those of the victim and the suspect.
• If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene (it does not prove they committed the crime).
• If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry.
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Example • A violent murder occurred.
• The forensics team retrieved a blood
sample from the crime scene.
• They prepared DNA profiles of the
blood sample, the victim and a suspect
as follows:
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Was the suspect at the crime scene?
Suspects Profile
Blood sample from crime scene
Victims profile
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Famous Cases
• Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence.
• He was arrested in 1986 for the rape and murder of two girls and was sentenced in 1988.
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Solving Medical Problems
DNA profiles can be used to determine whether a particular person is the parent of a child.
A childs paternity (father) and maternity(mother) can be determined.
This information can be used in • Paternity suits • Inheritance cases • Immigration cases
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Example: A Paternity Test
• By comparing the DNA profile of a
mother and her child it is possible to
identify DNA fragments in the child
which are absent from the mother and
must therefore have been inherited
from the biological father.
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Is this man the father of the child? Mother Child Man
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• Shown at the right are DNA "fingerprints" from a new mother (M, Fern LaPlante), her recently born child (C), and two possible fathers (F1, Ross and F2, Rick). As is to be expected, the mother and the child share a few DNA bands since they also share 50% of their genes but they each have bands that are not represented among the bands of the other.
• What do you think - was it Ross (F1) or was it Rick (F2)?
Paternity
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Gel Electrophoresis
• Electrophoresis - the migration of charged molecules in an electric field though a solution or solid support
• Various types – defined by support used
1. Paper – amino acids, small peptides
2. Polyacrylamide – Proteins, small DNA/RNA (<500bp)
3. Agarose – DNA/RNA
• Good preparative and analytical method
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• Detection 1. Dye e.g. ethidium bromide 2. Audioradiography 32P, 3. Blotting (see later) • Uses 1. Analytical- Can determine size of DNA
fragment, 2. Preparative – Can identify a specific fragment
based on size
Gel Electrophoresis
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Stages of DNA Profiling
• Agarose gel
electrophoresis. • Electrical current
carries negatively-charged DNA through gel towards positive (red) electrode
Power Supply
Buffer
Dyes
Agarose gel
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Theory of Electrophoresis The movement of a charged molecule subjected to an electric field is represented by the following equation:
V = Eq f
V: the velocity of the molecule
E: the electric field in volts/cm
q: the net charge on the molecule
f: frictional coefficient, which depend on the mass and shape of the molecule
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Agarose Gel electrophoresis
• DNA is placed at one end of a gel
• An electrical current is applied to the gel
• DNA molecules are negatively charged and move toward positive end of gel
• Smaller molecules move faster than larger ones
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1. Separation of charged molecules in an electric field.
2. Nucleic acids have 1 charged phosphate (- charge) per nucleotide. means constant chare to mass ratio. Separation based (mostly) on length: longer molecules move slower.
3. Done in a gel matrix to stabilize: agarose or acrylamide.
4. average run: 100 Volts across a 10 cm gel, run for 2 hours.
5. Stain with ethidium bromide: intercalates between DNA bases and fluoresces orange.
6. Run alongside standards of known sizes to get lengths
Gel Electrophoresis
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• Gel electrophoresis uses a cross-linked polymers (agarose) that contain various pores.
• Pores allow molecular sieving, where molecules e.g. DNA, can be separated based upon there mobility through the gel.
Gel Electrophoresis
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Mobility = Charge + Molecular Dimensions
• Charge per nucleic acid is constant
•This means separation is based upon length of the DNA molecules and this is how we can separate and identify DNA molecules.
Gel Electrophoresis
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Stages of DNA Profiling
DNA is separated on basis of size.
Notice the shorter fragments make it through the gel quicker than the longer fragments
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Stages of DNA Profiling • A radioactive
material is added which combines with the DNA fragments to produce a fluorescent image.
• A photographic copy of the DNA bands is obtained.
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Analysis of Stained Gel Determining restriction
fragment sizes
• Create standard curve using
DNA marker
• Measure distance traveled by
restriction fragments
• Determine size of DNA
fragments
• Identify the related samples
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• Linear DNA has a linear relationship to distance migration.
• If add molecular markers of known mass can calculate mass of our fragment by plotting a linear plot.
Gel Electrophoresis
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Molecular Weight Determination
Size (bp) Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0
100
1,000
10,000
100,000
0 5 10 15 20 25 30
Distance, mm
Siz
e, b
ase p
air
s
B
A
Fingerprinting Standard Curve: Semi-log
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Applications of Gel
Electrophoresis • Southern blot is produced when DNA on a
nitrocellulose blot is hybridized with a DNA probe.
• Northern blots are generated when RNA is hybridized with a complementary DNA probe produced by the reverse transcription of messenger RNA.
• A slightly different but related technique, known as a Western blot, involves separating proteins by gel electrophoresis and probing with labeled antibodies for specific proteins.
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ELECTROFORESIS
BLOTTING
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What is blotting?
Blots are techniques for transferring DNA , RNA and
proteins onto a carrier so they can be separated, and
often follows the use of a gel electrophoresis. The
Southern blot is used for transferring DNA, the Northern
blot for RNA and the western blot for PROTEIN.
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TYPES OF BLOTTING TECHNIQUES
Blotting technique
Southern Blot
It is used to detect DNA.
Northern Blot
It is used to detect RNA.
Western blot
It is used to detect protein.
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TYPES OF BLOTTING TECHNIQUES
Southern blot
(DNA)
Northern blot
(RNA)
Western blot
(Protein) Eastern blot
(???)
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SOUTHERN BLOTTING • Professor Sir Edwin Southern,
Professor of Biochemistry and Fellow of Trinity developed this method in 1975.
• Southern won the Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences he developed this procedure at Edinburgh University more than 30 years ago. The technique is known as DNA transfer or 'Southern blotting'
Professor Sir Edwin Southern
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• Southern blotting combines agarose gel electrophoresis
for size separation of DNA with methods to transfer the
size separated DNA to a filter membrane for probe
hybridization.
• The key to this method is Hybridization.
• Hybridization - Process of forming a double-stranded
DNA molecule between a single-stranded DNA probe
and a single-stranded target patient DNA.
SOUTHERN BLOTTING
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PRINCIPLE 1. The mixture of molecules is separated.
2. The molecules are immobilized on a matrix.
3. The probe is added to the matrix to bind to the
molecules.
4. Any unbound probes are then removed.
5. The place where the probe is connected corresponds
to the location of the immobilized target molecule.
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Capillary plotting apparatus
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Steps in southern blotting 1. Digest the DNA with an
appropriate restriction
enzyme.
2.The complex mixture of
fragments is subjected to gel
electrophoresis to separate the
fragments according to size.
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3.The restriction fragments present
in the gel are denatured with
alkali and transferred onto
4. a nitrocellulose filter or nylon
membrane by blotting.
• This procedure preserves the
distribution of the fragments in
the gel, creating a replica of the
gel on the filter.
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5.The filter is incubated under
hybridization conditions with
a specific radiolabeled DNA
probe.
• The probe hybridizes to the
complementary DNA
restriction fragment.
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6.Excess probe is washed away and the
probe bound to the filter is detected by
autoradiography, which reveals the
DNA fragment to which the probe
hybridized.
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APPLICATIONS • Southern blots are used in gene discovery , mapping,
evolution and development studies, diagnostics and
forensics (It is used for DNA fingerprinting, preparation
of RFLP maps)
• identification of the transferred genes in transgenic
individuals, etc.
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APPLICATIONS • Southern blots allow investigators to determine the
molecular weight of a restriction fragment and to
measure relative amounts in different samples.
• Southern blot is used to detect the presence of a
particular bit of DNA in a sample
• analyze the genetic patterns which appear in a person's
DNA.
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Northern Blotting Northern blotting is a technique for detection of specific
RNA sequences. Northern blotting was developed by
James Alwine and George Stark at Stanford University
(1979) and was named such by analogy to Southern
blotting
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Steps involved in Northern blotting
1. RNA is isolated from several
biological samples (e.g. various
tissues, various developmental
stages of same tissue etc.)
* RNA is more susceptible to
degradation than DNA.
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2. Sample’s are loaded on gel and
the RNA samples are separated
according to their size on an
agarose gel .
• The resulting gel following after
the electrophoresis run.
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3. The gel is then blotted
on a nylon membrane
or a nitrocellulose filter
paper by creating the
sandwich
arrangement.
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4. The membrane is placed in a dish
containing hybridization buffer
with a labeled probe.
• Thus, it will hybridize to the RNA
on the blot that corresponds to
the sequence of interest.
5. The membrane is washed to
remove unbound probe.
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6. The labeled probe is detected via
autoradiography or via a
chemiluminescence reaction (if a
chemically labeled probe is used). In
both cases this results in the
formation of a dark band on an X-
ray film.
• Now the expression patterns of the
sequence of interest in the different
samples can be compared.
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APPLICATIONS • A standard for the study of gene expression at the level of
mRNA (messenger RNA transcripts)
• Detection of mRNA transcript size
• Study RNA degradation
• Study RNA splicing
• Study RNA half-life
• Often used to confirm and check transgenic / knockout mice
(animals)
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Disadvantage of Nourthern plotting
1.The standard northern blot method is relatively less
sensitive than nuclease protection assays and RT-PCR
2. Detection with multiple probes is a problem
3. If RNA samples are even slightly degraded by RNases,
the quality of the data and quantitation of expression
is quite negatively affected.
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Western blotting • Western blotting (1981) is an Immunoblotting technique
which rely on the specificity of binding between a
protein of interest and a probe (antibody raised against
that particular protein) to allow detection of the protein
of interest in a mixture of many other similar molecules.
• The SDS PAGE technique is a prerequisite for Western
blotting .
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Steps in western blotting
1. A protein sample is subjected to
electrophoresis on an SDS-
polyacrylamide gel.
2. Electroblotting transfers the
separated proteins from the gel to
the surface of a nitrocellulose
membrane.
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3. The blot is incubated with a generic protein (such
as milk proteins or BSA) which binds to any
remaining sticky places on the nitrocellulose.
4. An antibody that is specific for the protein of
interest (the primary antibody - Ab1) is added to
the nitrocellulose sheet and reacts with the
antigen. Only the band containing the protein of
interest binds the antibody, forming a layer of
antibody molecules .
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5. After washing for removal of non-
specifically bound Ab1, second
antibody (Ab2)is added, which
specifically recognizes the Fc domain of
the primary antibody and binds it. Ab2
is radioactively labeled, or is covalently
linked to a reporter enzyme, which
allows to visualize the protein-Ab1-Ab2
complex.
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An example
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Application 1.The confirmatory HIV test
2.Western blot is also used as the definitive test for Bovine
spongiform encephalopathy (BSE(
3.Some forms of Lyme disease testing employ Western
blotting .
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Comparison of Southern, Northern, and Western blotting techniques
Southern blotting Northern blotting Western blotting
Molecule detected
DNA (ds) mRNA (ss) Protein
Gel electrophoresis
Agarose gel Formaldehyde agarose gel
Polyacrylamide gel
Gel pretreatment
Depurination, denaturation, and
neutralization
- -
Blotting method Capillary transfer Capillary transfer Electric transfer
Probes DNA Radioactive or nonradioactive
cDNA, cRNA Radioactive or nonradioactive
primary antibody
Detection system
Autoradiography Chemiluminescent
Colorimetric
Autoradiography Chemiluminescent
Colorimetric
Chemiluminescent Colorimetric
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THANK YOU 60