Method: Prehybridization of Repeat-containing Probes with Total Human DNA

4/18/1990

C. Helms

Purpose:

Unless clones derived from genomic human DNA are known to be single copy, they can be assumed to contain highly repetitive sequences such as Alu. Hybridizing a probe withrepeats to a human DNA Southern blot will result in a signal in each human lane that looks very much like the ethidium bromide stained pattern: a continuous smear with little or no additional signal from the unique sequences on the probe. To reduce or eliminate the lane background due to the repetitive sequences, we add a large amount of unlabeled human DNA (sonicated to an average size of 400-700 bp for highest efficiency) to the labeled probes immediately before the denaturing and hybridizing steps. The excess number of repeat sequences in the human genomic DNA competes with the probe sequences for those on the blot, effectively reducing the lane background by preventing the probe repeats from hybridizing to the blot. With some probes, this treatment is not sufficient to reduce the lane background. If this is the case, the first thing to try is prehybridization of the probe with the unlabeled human DNA. The probe hybridizes to the unlabeled human repeats before it ever sees the blots, and once added to the hybridization bag, only the unique sequences from the probe should be available for hybridization. If prehybridization does not solve the problem, try finding a restriction fragment which does not contain the repeat but does have unique sequences.

Time required:

a minimum of one hour.

Procedure:

1. Add 300 µl unlabeled sonicated human placental DNA to the labeled probe ( ~500 µl). If required for size marker detection, also add the appropriate amount of labeled lambda probe.
2. Denature by heating 10 minutes in a boiling water bath, then add the probe+ unlabeled human DNA mix to 10-20 ml prehybridization mix (in a 50 ml orange-cap tube). Mix thoroughly by inverting the tube several times. Rock the tube at the incubation temperature (usually 50 degrees C) for 30 minutes to overnight.
3. After the prehybridization period, add the probe + prehyb mix to the hybridization bag, and proceed with the hybridization as usual.

References: none