Chapter 6: Hybridisation Using Oligonucleotide Probes - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

HYBRIDISATION USING OLIGONUCLEOTIDE PROBES
The Hybridisation conditions outlined in Chapter 3 are generally applicable to probes
derived by cloning, or by gene amplification reactions ranging in size from 100bp to
several hundred base pairs. The Hybridisation conditions of synthetic oligonucleotide
probes, however, are rather more problematical.
There are two types of oligonucleotide probes: -
1. A single oligonucleotide of a defined sequence, derived from the nucleic acid
sequence of the target gene.
2. A pool of oligonucleotides of degenerate sequences, which are derived from the
amino acid sequence of a region of the gene product of interest. Due to the
degeneracy of the genetic code (i.e. most amino acids are specified by more than
one triplet codon) any particular sequence of amino acids will be specified by a
number of different oligonucleotides.
Because the Tm of synthetic oligonucleotides is much lower than for longer probes,
the stringency of Hybridisation and washing procedures must be reduced and
adjusted according to the base composition of the probe. An approximate value for
the Tm of an oligonucleotide probe has been derived by Wallace et al (1979): -
Hybridisations are carried out at 5°C below this temperature. For a degenerate pool
of oligonucleotides, Hybridisation is carried out at 5°C below the lowest Tm for the
set of sequences.
1. Prehybridise the membrane in oligonucleotide prehybridisation buffer (6 x SSPE
(SSC), 10 x Denhardt's reagent, 50µg/ml denatured salmon sperm DNA) at the
Hybridisation temperature for at least one hour.
2. Add the radio-labelled probe solution to the preHybridisation buffer. Alternatively,
remove a small amount of preHybridisation buffer, add the probe to this and
replace this solution in the Hybridisation vessel. Some researchers may prefer to
use fresh Hybridisation buffer.
3. Hybridise with agitation, or by rotating in bottles, for approximately 12 hours at the
Hybridisation temperature.
© Thermo Scientific, May 2003. Issue 7
CHAPTER 6
HYBRIDISATION GUIDE
Tm (°C) = 2 x (number of AT base pairs) +
4 x (numbers of GC base pairs).
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