Thermo Scientific Shake 'n Stack 6244, Operating And Maintenance Manual

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© Thermo
Scientific
, May 2003. Issue 7 18
CHAPTER 4
HYBRIDISATION GUIDE
NUCLEIC ACID PROBES
There are now several types of nucleic acid probe available to researchers and a wide
variety of radio-labelling techniques for labelling these probes.
For convenience, the types of nucleic acid probe may be classified as follows: -
1. Double-stranded DNA probes
2. Single-stranded DNA probes
3. RNA probes
4. Synthetic oligonucleotides
Double-Stranded DNA Probes
Any suitable DNA molecules (cloned or uncloned) can be used as a Hybridisation probe,
for example, insert fragments from a DNA library may be excised from plasmids or
bacteriophages by restriction enzyme digest and then labelled for Hybridisation. Another
possibility is to use the Polymerase Chain Reaction to synthesize copies of the region of
interest, which may be radio-labelled during the amplification reaction, itself, or the final
product labelled post-amplification. Labelling of double-stranded DNA may be carried out
by NICK TRANSLATION OR PRIMER EXTENSION. Commercially available kits enable
these techniques to be carried out simply and efficiently resulting in probes of high
specific activity, often requiring no further purification before adding to the Hybridisation
buffer.
Removal of unincorporated nucleotides may be advantageous in reducing background.
This may be simply carried out using Thermo Recovery kits.
Single-Stranded DNA Probes
Single-stranded DNA probes may be synthesized from mRNA using Reverse
Transcriptase, or may be derived from fragments cloned into specialised M13 or
phagemid vectors, which contain the origin of replication of a single-stranded DNA
bacteriophage. Synthesis of the DNA strand complementary to the region of interest
incorporating a
32
P labelled dNTP results in a single-stranded radio labelled probe
molecule, which is then separated from the unlabelled template by gel electrophoresis
(the fragment may then be extracted from the gel using Thermo Recovery kits). This
technique eliminates the possibility of re-association of complementary strands, which
can occur with double-stranded DNA probes.