A technique used to identify and locate mRNA
sequences that are complementary to a piece of DNA
called a probe.
As shown in the diagram, the procedure involves four steps:
- RNA (either total RNA or just mRNA) is
separated by gel electrophoresis,
usually an agarose gel. Because there are so many different RNA molecules
on the gel, it usually appears as a smear rather than discrete bands.
- The RNA is transfered to a sheet of special blotting paper called
nitrocellulose, though other types of paper, or membranes, can be used.
The RNA molecules retain the same pattern of separation they had on
- The blot is incubated with a probe which is single-stranded DNA. This
probe will form base pairs with its
complementary RNA sequence and bind to form a double-stranded RNA-DNA
molecule. The probe cannot be seen but it is either radioactive or has
an enzyme bound to it (e.g. alkaline phosphatase or horseradish peroxidase).
- The location of the probe is revealed by incubating it with a colorless
substrate that the attached enzyme converts to a colored product that
can be seen or gives off light which will expose X-ray film. If the
probe was labeled with radioactivity, it can expose X-ray film directly.
Source: Department of Biology, Davidson College, NC