HYBRIDIZATION

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HYBRIDIZATION
By
Dr. Emad AbdElhameed Morad
Lecturer of Medical Microbiology and Immunology
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• Denaturation or melting Separation of the two
  DNA strands from each other. Melting could be
  done by either
• Heat (90-100 c)
• Alkaline pH
• Low salt concentration
• Renaturation or annealing Reassociation of the
  two DNA strands to form a double helix. Annealing
  could be done by
• Lowering the temperature
• Neutralization of pH
• Raising the salt concentration

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• Nucleic acid probe
• A short known sequence of single stranded DNA or
  RNA designed to hybridize with the target nucleic
  acid.
• Nucleic acid probes (known) are thus used in
  identification of microbes (unknown).
• The probes are labeled to facilitate their
  detection following hybridization.

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Hybridization based assays
Solid phase hybridization
Solution phase hybridization
Dot blot
In situ hybridization
Blotting techniques
FISH
Northern
Southern
Western
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Solid phase hybridization
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Dot blot

• The bacterial cells are lysed to liberate DNA.
• The DNA is treated by alkali or heat to separate
  the two strands.
• The DNA strands are attached to nitrocellulose
  membrane.
• Labeled probe is then applied to the membrane.
• If the probe is complementary to the test DNA, it
  will form a hybrid duplex with the test DNA and
  remain on the membrane after washing.
• Formation of hybrid DNA is proved by detecting
  the label conjugated to the probe.

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Stringency of hybridization

• The temperature, salt concentration and sequence
  homology forms the conditions of stringency for
  hybridization.
• Stringency increases as the salt concentration
  decreases and temperature increases.
• More stringent conditions permit hybridization of
  only highly homologous sequence.
• A single base mismatch in a stretch of 20 bases
  may prevent hybridization under the most
  stringent conditions.
• RNA-RNA duplexes are more stable than RNA-DNA
  duplexes, which are more stable than DNA-DNA
  duplexes.

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Labels of probes

• Labeling can be done using radioactive or
  non-radioactive labels.
• The common radioactive isotopes used for labeling
  include P 32, S 35, I 125.
• Once hybridized, the labeled probes can be
  detected by scintillation counter or on X-ray
  autoradiography.
• Disadvantages include
• Higher expense
• Difficulty in handling
• Health hazard

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Radioactive labels of probes
The upper lane is Ve
The lower lane is -Ve
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• The non-radioactive labels include biotin,
  digoxygenin , acridinium esters.
• Biotin binds specifically to avidin. Avidin is
  tagged with enzymes. Addition of substrate
  results in production of coloured product,
  signaling the positive hybridization reaction.
• Avidin may be conjugated with fluorescein.
  Hybridization is observed for fluorescence using
  UVL.
• Detection of hybridization may be done by using
  fluorescein conjugated antibodies against biotin.

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• Digoxygenin labeled probes are detected by enzyme
  labeled anti-digoxygenin antibody and then using
  substrate to detect it.
• Probes can also be labeled with acridinium ester.
  
• Detection is achieved by the addition of hydrogen
  peroxide hydroxide, which results in hydrolysis
  of the ester linkage.
• The light that is produced in this reaction is
  detected using a chemiluminometer.

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Non- radioactive labels of probes
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In situ hybridization

• Hybridization process performed on intact tissues
  or cells fixed to a glass microscopic slide.
• Fluorescence in situ hybridization (FISH)
• In situ hybridization (ISH) using probe labeled
  with a fluorescent dye.
• Examination is done using fluorescent microscope.

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GOOD LUCK