Components

• What are the components of Nucleic Acid Hybridization?

  Probe

  Target
• It is a component of Nucleic Acid Hybridization that is a single-stranded fragment of nicleic acid attached to a signal-producing moiety to identify sequence/s of interest.


  Probe
• The Probe is labelled with?

  Radioisotope

  Fluorescent Molecule
• It is a component of Nucleic Acid Hybridization that is a denature DNA or RNA that has good complementarity to the probe for identification.

  Target
• The Target is bound to?

  Nylon Membrane

  Another solid surface
• Early methods of production of a DNA probe.

  Cloning into a plasmid

  Isolation by RE digestion

  Gel purification

  Labelling

  Denaturation(heating alone or with formamide)
• New methods of production of a DNA probe.

  Isolation from viral genomes

  In vitro organic synthesis (short, oligometric probes)

  PCR (for production of large amounts)
• Longer probes are more?

  Specific
• Shorter probes are better for?

  Mutational Analyses (can detect single base changes)
• What matters for both DNA and RNA probes?

  Sequence
• It is a probe type that is made by in vitro transcription from DNA (from plasmid template or PCR amplification). It has equal or greater binding affinity, and is more sensitive.

  RNA
• Production of RNA probes

  1. Cloning into plasmid
  2. Linearization of recombinant vector
  3. Transcription from promoter Normal - Southern Antisense - Northern
  4. Labeling
  5. Removal of DNA template
• Must be done so the probe generates a detectable signal.

  Labelling the probe
• Types of probe labeling

  End labeling

  Nick translation

  Random labeling
• Labeled molecules added to the end using terminal transferase or T4 polynucleotide kinase.

  End Labeling
• Labeled nucleotides added into single-stranded breaks that become substrates for DNA polymerase to extend the nick.

  Nick Translation
• Generation of new single-stranded version of the probe with labeled nucleotides.

  Random Priming
• They are no longer favored because they are hazardous to humans and the environment. their half-lives are short and thus, experiments must be done quickly.

  Radioisotopes
• Introduction of nucleotides with radioactive phosphorus (32P), sulfur (35S), or nitrogen to the probe. Unbound probes get washed off. Blots are exposed to light-sensitive film to detect the hybridized fragments.
• Most common non radioactive labels

  Biotin

  Digoxygenin
• They are based on indirect detection of tagged nucleotides incorporated or added to the probe.

  Non-Radioactive Labels
• Detection of Non-Radioactive Labels

  1. Washing off of unbound probe
  2. Antidioxygenin (dioxygenin) or Streptavidin (biotin) are conjugated to alkaline phosphatase or horseradish peroxidase (HRP)
  3. Washing off of unbound conjugate
  4. Bathing in substrate (dioxetane or tetrazolium dye)