Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Phosphorylation of DNA Molecules with Protruding 5-Hydroxyl Termini

(Protocol summary only for purposes of this preview site)

The removal of 5 phosphates from nucleic acids with phosphatases and their readdition in radiolabeled form by bacteriophage T4 polynucleotide kinase is a widely used technique for generating 32P-labeled probes (see the box Labeling the 5 Termini of DNA with Bacteriophage T4 Polynucleotide Kinase, below). When the reaction is performed efficiently, 4050 of the protruding 5 termini in the reaction becomes radiolabeled (Berkner and Folk 1977). However, the specific activity of the resulting probes is not as high as that obtained by other radiolabeling methods because only one radioactive atom is introduced per DNA molecule. Nevertheless, the availability of [-32P]ATP with specific activities in the 30007000 Ci/mmol range allows the synthesis of probes suitable for many purposes, including the following:

  • as primers in primer-extension experiments to map the 5 ends of mRNAs
  • as substrates in the analysis of RNA structure by nuclease S1


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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