DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 32

Probes to identify nucleic acids on membranes
To identify a particular nucleic acid (whether DNA or RNA) on a mem-
brane, probes are used. A probe is a general name for a substance that
is used to specifically identify a macromolecule. It can be a chemical,
an antibody, a protein, or a fragment of DNA.
For identifying nucleic acids on membranes, DNA probes are often
used. These probes are labeled by one of several methods with either a
radioactive isotope, a dye or fluorescent compound, or an enzyme. The
probes are hybridized (annealed) to nucleic acids on the membrane. If a
complementary nucleic acid is present, the probe anneals, and its pres-
ence can be detected by a method suited to the detection of the probe,
depending on the labeling method. The probe must be single stranded in
order to hybridize. As a result, labeled double stranded DNA fragments
must be denatured prior to hybridization. Single stranded probes can
also be synthesized using the appropriate vector (see below).
After hybridization reactions, membranes are washed in solutions
containing formamide, SSC (a salt solution containing sodium chloride
and sodium citrate) and SDS (sodium dodecyl sulfate). By reducing
the concentration of SSC and increasing the temperature at which the
membrane is washed, only highly complementary segments of nucleic
acids will remain annealed to the probe. This is generally referred to as
increasing the stringency between probe and target sequence.
Autoradiography (exposing the blot to x-ray film) is typically used to
visualize the hybridization of a radioactive probe to the membrane. Al-
ternatively, the hybridized probe can be identified and quantitated by
phosphorimaging. For probes that have been labeled with fluorescent
dyes or enzymes, colorimetric methods that rely on chemiluminescence
can also be used (see Chapter 4).
C. DNA sequencing
Introduction
DNA sequencing is, as the name implies, a method that reveals the
linear sequence of nucleic acids within a stretch of DNA. DNA is com-
posed of four nucleotide bases: adenine (A), cytosine (C), guanine (G),
and thymine (T). The base present at a particular position in a DNA
sequence is identified by the presence of a product of a chemical or
enzymatic reaction in a gel. The ability to sequence DNA was a major
advance for a number of reasons. First, it allows for the prediction of a
potential open reading frame (ORF) for genes within a genomic DNA
sequence. An ORF is the sequence that is transcribed and translated