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8/2/2019 Lec 5 - Dna Hybridization
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CHAPTER 5
DNA HYBRIDIZATION
-INTRODUCTION
-DNA PROBE, LABELING, SIGNAL
MISS NUR SHALENA SOFIAN
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INTRODUCTION
DNA hybridization ability of ssDNA of onestrand to form dsDNA of another ssDNA bycomplementary sequences
Probe is used to identify a desired cloneamong thousands of irrelevant ones :
a. polynucleotides / oligonucleotides - Must
have a probe to screen the libraryb. antibodies - Must have antibodies specificfor your protein
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ANTIBODY PROBE
E.g. gt11 vector for making and screening cDNA
libraries
Group of clones are screen via expression of the
right protein therefore need antiserum directed
against protein of interest
When cDNA inserts are plated, proteins released
by each clone are blotted onto nitrocellulose Proteins that have transferred onto nitrocellulose
can be probed with antiserum
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Antibody bindstightly to protein andlabels thecorresponding spotwhen detected byautoradiography orphosphorimaging
The right clone canthen be picked fromthe master plate
Antiserum is mixtureof antibodies thatcan react withseveral different
parts of protein
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POLYNUCLEOTIDE PROBE
Using homologous gene from another
organism to probe for gene of interest
Lowering stringency of hybridization
conditions so it can tolerate mismatches in
base sequence between the probe and the
cloned gene e.g. high temperature, organic
solvents, salt concentrations
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DNA Hybridization
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Labeled Tracers
Radioactive tracers allow small quantities of
substances to be detected
E.g. RNA quantities of less than pg not suitable
using UV light absorption or staining with dyes
Most widely tracers used:
- autoradiography
- phosphorimaging
- liquid scintillation
- scintillation counting
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a. Autoradiography
Detecting radioactive compounds with photographicemulsion x-ray film
Radioactive DNA fragments underwentelectrophoresis. Later the gel is placed in contact withx-ray film, leaves in dark
Radioactive emissions from the bands of DNAproducing dark bands
Estimating the intensity of the bands by scanning withdensitometer
Measures the absorbance of light by the sample themore intensify the bands, the more it will most light
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Analyzing in autoradiography alongsidedensitometry and molecular weight determination,as well as an overlay for annotating gel images. Thepeaks produced proportional to the darkness of the
corresponding bands on autoradiograph
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b. Phosphorimaging
Responding to radioactivity far more linear thanof x-ray film
A radioactive sample (blotting DNA bands thathave been hybridized with labeled probes)
Sample is placed in contact with phosphorimagerplate, absorbing -rays
The rays excite the molecules until phorimagerscans the plate with laser
-rays energy is released, monitored bycomputerized detector
Computer converts energy to an image in false
color
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Nucleosome formation during DNA
repair inXenopus extracts. The picture is
a phosphorImage of a native
polyacrylamide gel showing DNA and
nucleosome complexes before and after
repair.
Kosmoski, J. V, Ackerman, E. J. and
Smerdon, M. J. (2001). DNA repair of a
single photoproduct in a design
nucleosome. PNAS. 98
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a. DNA Hybridization: Simple Dot Blot
To detect biomolecules
Represents a simplification of the Northern blot,Southern blot, or Western blot methods
The biomolecules to be detected are not firstseparated by chromatography. Instead, a mixturecontaining the molecule to be detected is applieddirectly on a membrane as a dot
Detect by either nucleotide probes (for aNorthern blot and Southern blot) or antibodies(for a Western blot)
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However, it offers no information on the size
of the target biomolecule
If two molecules of different sizes aredetected, they will still appear as a single dot
Dot blots therefore can only confirm the
presence or absence of a biomolecule(s)which can be detected by the DNA probes or
the antibody