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Wednesday, May 1, 2013

Labelling of DNA / RNA


Labelling of DNA & RNA can be done using:

  1. Radioactive isotopes
  2. Non-Radioactive isotopes
commonly used radioactive isotopes are : 125I, 14C 15N, 35S, 32P, etc.
Labelling can be done using 3 methods
  1. Nick Translation
  2. End Labelling
  3. Random Primer Labelling

Nick Translation

  • Rapid, easy and inexpensive method.
  • Amount of DNase  I added is critical
  • Reaction stopped using EDTA.
  • Purification can be done either by Column chromatography or by Gel Extraction.
Nick Translation

End Labelling

  • End labelling can be done by two ways: 3’ End Labelling and 5’ End Labelling.
End Labelling
  • The Selected DNA is digested with restriction enzymes to generate sticky end.
radioaactive end labelling

Disadvantages of Radioactive Isotope Labelling

  • It is hazardous
  • Require long exposure times
  • Costly
  • Instability due to the half life period of radioactive isotopes.
Non Radioactive Isotope DNA Labelling
  • Safe.
  • Stable.
  • Economical.
  • More stable compared to radioactive isotope labelling.
There are two methods of Non radioactive isotope labelling
  1. Labelling with Biotinylated nucleotide
  2. Labelling with HRP conjugate
nonradioactive labelling

Disadvantages of Non Radioactive Isotope Labelling

  • Less Sensitive