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.

 

  See the Movie about
Probe Labeling

Read the Movie Text

 

 

 

Digoxigenin

The digoxygenin group is the label used in the Southern hybridisation step in the Simple Cloning Lab.
Below, the molecular structure of Dig-dUTP is shown.
Dig-dUTP is recognised by DNA polymerase as dTTP, and will be incorporated in the newly synthesised DNA (during the probe labeling reaction), when the polymerase reads an adenine nucleotide in the template. So, highly antigenic digoxigenin will be present in the new DNA.

 

Digoxigenin can be detected by antibodies, conjugated to the enzyme Alkaline Phosphatase. This enzyme can subsequently be used in a staining protocol, to visualise the localisation of labeled DNA.