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Nucleic Acid Hybridization

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Nucleic Acid Hybridization. Nucleic acid hybridization is a fundamental tool in ... Benton-Davis. Bacterial colony blotting. Grunstein-Hogness. Slot/Dot blotting ... – PowerPoint PPT presentation

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Title: 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
  • 5
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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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06_10.jpg
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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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06_12_2.jpg
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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
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