http://insilico.ehu.es/tm.php

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• Labelling probes and primers
• In the cases of Northern and Southern blots
  probes are pieces of single stranded DNA that are
  complimentary to the single stranded target on
  the membrane.
• Short oligonucleotides or primers are used for
  labeling sections for light, fluorescent or
  electron microscopy and also in amplification
  reactions
• How are probes made?
• synthetically
• cDNA derived from RNA by using reverse
  transcriptase
• Isolated from genomic DNA using PCR or
  restriction digest followed by PCR
• Isolated from many copies of plasmids following
  digestion and gel isolation
• Isolated from a specialized viral vector that
  makes single stranded DNA
• Probes and primers vary in size from 20 to 1000s
  of bases
• Probes for Southerns tend to be at least 100bp
  long

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• Probes for a blot must be tagged so we can
  detect them when bound to the target on the
  membrane
• Common labels include radio-isotopes e.g. 32P,
  fluorescent molecules e.g. acridine orange, and
  colour generating enzymes e.g. alkaline
  phosphatase
• When the probe is generated one or more
  nucleotides needs to be labeled
• For detection of probes in electron microscopy
  the label needs to be electron dense e.g. gold

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• Methods of labeling probes
• 1. 5 end labeling
• Use an enzyme such as T4 polynucleotide kinase
  to catalyse the transfer of a gamma phosphate
  from ATP to the 5OH of the probe. Works for ss
  and ds DNA
• A -P-P-P
• 5OH-----------------3OH ? 5
  P------------------3OH ADP

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• 2. 3 end labeling
• Use TdT to add homopolymer extensions at the 3OH
  of a probe. Works for ss and ds DNA

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• 3. PCR based labeling
• Probe is amplified by PCR in the presence of
  labeled nucleotides, which are incorporated into
  newly amplified DNA
• this method generates more probe and labels it,
  unlike most other methods which require large
  quantities of DNA to label.

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• 4. Nick translation based labeling
• Dnase 1 nicks the DNA (cuts phosphodiester bonds)
• DNA polymerase (with a 5 to 3 exonuc act)
  replaces nucleotides with new dNTPs, one or more
  of which is labeled.

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• Using Biotin as a tag
• Biotin is a naturally occurring B vitamin,
  normally used in energy metabolism. Avidin and
  Strepavidin are proteins that normally act as B
  vitamin scavengers and bind Biotin. Avidin and
  Strepavidin bind with a very high affinity and
  we can use this to detect biotin labeled probes.
  The avidin or strepavidin can be attached to an
  enzyme or a fluorescent label.
• This gives you a choice as to how you will
  visualize your probe. Biotin can be incorporated
  as biotinylated dNTPs in a PCR reaction or end
  labeling or added as biotinylated dNTPs in a nick
  translation reaction.
• The required visualization agent, fluorescence,
  gold, enzyme is then added bound to strepavidin
  and binds to the biotinylated nucleotides.

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• The protein recognizing the group can be a
  specific antibody, e.g. the dioxygenin system or
  any other ligand that has a very high affinity
  for a specific group e.g. Biotin-strepavidin.
• The marker can be detected in various ways e.g.
• if it carries a fluorescent dye it can be
  detected in a fluorimetric assay
• it can be an enzyme such as alkaline phosphatase
  which can be coupled with an enzyme assay
  yielding a product that can be measured
  colorimetrically

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