Probe Labeling

Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR

Digoxygenin

Specific sequences in the restriction pattern on a Southern blot can then be identified by hybridization with a DNA probe. A probe is a piece of DNA identical (or very similar) to a sequence of interest. In order to locate a specific DNA sequence by hybridization, the probe is labeled with a reporter group.

The Klenow fragment of E.coli DNA polymerase is used to make a labeled probe. This so-called Klenow enzyme is a truncated version of polymerase I: it lacks the 5’ to 3’ exonuclease activity. It can synthesize DNA complementary to a DNA template in a 5’ to 3’ direction, provided it is given a primer with a free 3’-OH, and dNTP's. A DNA fragment, often purified from agarose gel or a cleaned-up PCR product, is chosen to act as a template in the polymerase reaction. The duplex DNA is first denatured by boiling in the presence of a mixture of all possible hexanucleotides (small, 6 nucleotides long DNA molecules with a random sequence). These so-called random primers anneal at positions of complernentarity along the single strands and act as starting points for the Klenow polymerase to synthesize a complementary strand. By using digoxygenin-labeled dNTPs in the reaction mixture, the newly synthesized strands will be labeled with digoxigenin. Eventually, a mixture of randomly sized labeled copy fragments of both strands are present.
The labeled DNA can be used as a probe in a blot hybridization experiment. Labeled DNA can be located on a blot by using antibodies against digoxigenine.

note: a labeled probe can detect a band, even when it contains only a small part of the sequence present in that band!

note:
Klenow polymerase is also used in the blunting protocol for the construction of pGTΔB.