lec 5 - dna hybridization

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

lec 5 - dna hybridization

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