Nucleic Acid Hybridization

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Nucleic Acid Hybridization

• Nucleic acid hybridization is a fundamental tool
  in molecular genetics which takes advantage of
  the ability of individual single-stranded nucleic
  acid molecules to form double stranded molecules
  (that is, to hybridize to each other)

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Standard nucleic acid hybridization assays

• A labeled nucleic acid - a probe - to identify
• related DNA or RNA molecules
• Complex mixture of unlabeled nucleic acid
• molecules- the target
• Base complementarity with a high degree of
• similarity between the probe and the target.

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Probes

• DNA labelling
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• Uniform labeling
• Nick translation
• Random primer
• PCR-mediated labeling
• RNA labelling
• In vitro transcription of a cloned DNA insert
• Different probes
• Radioactive labeling or isotopic labeling
• Nonradioactive labeling or nonisotopic labeling

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Kinase end-labeling of oligonucleotides
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Fill-in end labeling
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Nick translation
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Random primed labeling
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Riboprobes
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Characteristics of radioisotopes commonly used
for labeling DNA and RNA probes

• Radioisotope Half-life Decay-type Energy
  of emission
• 3H 12.4 years b- 0.019 MeV
• 32P 14.3 days b- 1.710 MeV
• 33P 25.5 days b- 0.248 MeV
• 35S 87.4 days b- 0.167 MeV

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Nonisotopic labeling and detection

• The use of nonradioactive labels has several
  advantages
• safety
• higher stability of a probe
• efficiency of the labeling reaction
• detection in situ
• less time taken to detect signal
• Major types
• Direct nonisotopic labeling (ex. nt labeled with
  a fluorophore)
• Indirect nonisotopic labeling (ex.
  biotin.-streptavidin system)

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Structure of fluorophores
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Structure of digoxigenin-modified nucleotides
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Indirect nonisotopic labeling
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Nucleic acid hybridization- formation of
heteroduplexes
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Ultraviolet absortion spectrum of DNA
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Denaturation of DNA results in an increase of
optical density
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Melting curve of a specific DNA sequence
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Factors affecting Tm of nucleic acid hybrids

• Destabilizing agents (ex. formamide, urea)
• Ionic strenght
• Base composition (G/C, repetitive DNA)
• Mismatched base pairs
• Duplex lenght
• Different equations for calculating Tm for
• DNA-DNA hybrids
• DNA-RNA hybrids
• RNA-RNA hybrids
• Oligonucleotide probes

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Factors affecting the hybridization for nucleic
acids in solution (annealing)

• Temperature
• Ionic strenght
• Destabilizing agents
• Mismatched base pairs
• Duplex lenght
• Viscosity
• Probe complexity
• Base composition
• pH

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Stringency
High temperature
Low salt concentration
High denaturant concentration
Sequence lenght
Tm
High strigency
Perfect match complementary sequences
Perfect match non-complementary sequences
Sequence G/C content
Low strigency
Low temperature
High salt concentration
Low denaturant concentration
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Filter hybridization methods
Bacteriophage blotting Benton-Davis
Filter hybridization techniques
Bacterial colony blotting Grunstein-Hogness
Slot/Dot blotting
Northern analysis Southern analysis
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Filters or Membranes

• Nitrocellulose
• Nylon
• Positive charged nylon (hybond)
• PVDF (hydrophobic polyvinylidene difloride)
• Different properties
• Binding capacity (mg nucleic acids/cm2)
• Tensile strenght
• Mode of nucleic acid attachment
• Lower size limit for efficient nucleic acid
  retention

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Principles of Southern blot
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Southern Blotting Apparatus
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Depurination/Denaturation of DNA
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Typical hybridization solution

• High salt solution (SSC or SSPE)
• Blocking agent (Denhardts, salmon sperm DNA,
  yeast tRNA)
• SDS

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Southern Applications

• Detection of DNA rearrangements and deletions
  found in several diseases
• Identification of structural genes (related in
  the same species (paralogs) or in different
  species (orthologs))
• Construction of restriction maps

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Southern applications- example
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Colony blot hybridization-1
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Colony blot hybridization-2
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Colony blot hybridization- example