10 Hybridization Methods (Southern and Northern Blotting)
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DNA blot hybridization
There are two ways to blot nucleic acids:
• Direct spotting of DNA or RNA solutions on to nitrocellulose or nylon
filter membranes (dot or slot blot), and
• Transfer of DNA or RNA to filter membranes after size fractionation by
agarose gel electrophoresis (Southern or Northern blot). DNA is digested
with restriction enzymes prior to electrophoresis.
RNA blot hybridzation
RNA blot hybridization is a technique that involves the detection of specific
target sequences within total RNA or mRNA that has been immobilized to a
filter membrane. The subprotocols 4, 5 and 6 describe direct spotting of
RNA on to a membrane filter (dot blot).
Subprotocol 1
Preparation of Dot Blots for DNA
Dot blots are used to determine the presence or absence of specific sequences within the target DNA. In comparison with standard samples,
the amount of target DNA may also be quantified. It is not possible, however, to determine the number and sizes of bands that belong to the target
sequence. To achieve this, Southern blotting is the method of choice.
DNA dot blot hybridization has two components:
• preparation of DNA blots (filters), and
• hybridization of the filter to specific probes (see Subprotocol 3).
For the preparation of filters, commercial dot blot or slot blot manifolds
may be used. In this case, follow the manufacturer's instructions. A simple
and reliable protocol for direct spotting of DNA on to the membrane is
given in the following.