Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

Chapter 8 How to Obtain a Clone of a Specific Gene 143
Figure 8.18
B B B B
B
Cytochrome c gene Other genes
B
B
B B
A long cloned DNA fragment may contain several genes in addition to the one in which we are interested. B = BamHI
restriction site.
(a) Electrophorese BamHI-restricted DNA
(b) Southern transfer
Buffer
Wick
(c) Result of hybridization probing
Support
Positive signal
BamHI fragments
Paper towels
Nitrocellulose or
nylon membrane
Gel
Figure 8.19
Southern hybridization.
that we are not interested in are also present in this insert (Figure 8.18). The method
called Southern hybridization enables the individual restriction fragment containing the
cytochrome c gene to be identified.
The first step in using Southern hybridization for this purpose would be to digest the
clone with BamHI and then separate the restriction fragments by electrophoresis in an
agarose gel (Figure 8.19a). The aim is to use the oligonucleotide probe for cytochrome
c to identify the fragment that contains the gene. This can be attempted while the restriction
fragments are still contained in the electrophoresis gel, but the results are usually
not very good, as the gel matrix causes a lot of spurious background hybridization
that obscures the specific hybridization signal. Instead, the DNA bands in the agarose
gel are transferred to a nitrocellulose or nylon membrane, providing a much “cleaner”
environment for the hybridization experiment.
Transfer of DNA bands from an agarose gel to a membrane makes use of the technique
perfected in 1975 by Professor E.M. Southern and referred to as Southern transfer.
The membrane is placed on the gel, and buffer allowed to soak through, carrying
the DNA from the gel to the membrane where the DNA is bound. Sophisticated pieces
of apparatus can be purchased to assist this process, but many molecular biologists prefer
a homemade set-up incorporating a lot of paper towels and considerable balancing