GENE PROBES
Gene probes are single-stranded DNA or RNA.
Gene probes hybridize to a target DNA or RNA sequence.
Probe and target base sequences must be similar to each other but, depending on conditions, do not
necessarily have to be exactly identical, i.e. theircomplementarity is not always 100%.
Gene probes must be labelled otherwise the hybridization cannot be detected.
Gene probes are used in various blotting and in situ techniques for the detection of nucleic acid
sequences. In medicine they can help in the identification of microorganisms and the diagnosis of
infectious, inherited and other diseases.
Radiolabels
Probe nucleic acid can be labelled using radioactive isotopes, e.g. 32P, 35S, 125I, 3H.
Detection is by autoradiography or Geiger-Muller counters.
Radiolabelled probes used to be the most common type but are less popular today because of safety
considerations. However, radiolabelled probes are the most sensitive, e.g. 32P labelled probes can detect
single-copy genes in only 0.5 mg of DNA. High sensitivity means that low concentrations of probe-target
hybrid can be detected.
Non-radioactive labels
These are safer than radiolabels and do not require dedicated rooms, glassware and equipment or staff
monitoring, etc. but they are not generally as sensitive.
Some examples:
Biotin This label can be detected using avidin or streptavidin which have high affinities for biotin.
Enzymes The enzyme is attached to the probe and its presence usually detected by reaction with a
substrate that changes colour. Used in this way the enzyme is sometimes referred to as a "reporter group".
Examples of enzymes used include alkaline phosphatase and horseradish peroxidase.
Chemiluminescence In this method chemiluminescent chemicals attached to the probe are detected by
their light emission using a luminometer.
Fluorescence Chemicals attached to probe fluoresce under UV light. This type of label is especially
useful for the direct examination of microbiological or cytological specimens under the microscope - a
technique known as fluorescent in situ hybridization (FISH).
Antibodies An antigenic group is coupled to the probe and its presence detected using specific
antibodies. Also, monoclonal antibodies have been developed that will recognize DNA-RNA hybrids. The
antibodies themselves have to be labelled, e.g. using an enzyme.
ABELLING METHODS
Whatever label is used, it has to be attached to, or incorporated into, the nucleic acid probe.
Here are 5 methods of labelling probes (for more details of each click on the underlined link):
1. Nick translation
2. Primer extension
3. RNA polymerase
4. End labelling
5. Direct labelling
BRINGING PROBE AND TARGET TOGETHER
Probe and target sequence hybridize with each other but how this is brought about can vary. There are 4
main formats:
1. Solid support
Target (usually) is bound to a solid support such as a microtitre tray or filter membrane. Probe is
added in solution and binds to target (if present) on solid support. After washing to remove
unbound probe, hybridization is detected on the solid support using whatever method is
appropriate for the probe label, e.g. for a radiolabelled probe and a filter membrane
autoradiography could be used; for an enzyme labelled probe and a microtitre tray the colour
change of a substrate could be measured using an ELISA plate reader.
2. In solution
Both the probe and the target are in solution. Because both are free to move, the chances of
reaction are maximized and, therefore, this format is generally faster than others.
3. In situ
In this format probe solution is added to fixed tissues, sections or smears which are then usually
examined under the microscope. The probe label, e.g. a fluorescent marker, produces a visible
change in the specimen if the target sequence is present and hybridization has occurred. However,
the sensitivity may be low if the amount of target nucleic acid present in the specimen is low. This
can be used for the gene mapping of chromosomes, and for the detection of microorganisms in
specimens.
4. Southern & Northern blots
After size fractionation of nucleic acids by electrophoresis, they are transferred to a filter
membrane which is then probed. The presence of target is confirmed by detection of probe on the
filter membrane, e.g. radiolabelled probe can be detected by autoradiography and the location of
the target sequence in the bands in the original gel determined.
Southern blots are used for DNA analysis.
Northern blots blots are used for RNA analysis.
USES OF GENE PROBES
Probes hybridize to complementary DNA or RNA sequences and have several uses:
1. Southern blots
Detection of gel-fractionated DNA molecules transferred to a membrane. This includes restriction fragment
length polymorphism (RFLP) analysis.
2. Northern blots
As above but used for RNA.
3. Dot blots
Detection of unfractionated nucleic acid immobilized on a membrane.
4. Colony and plaque blots
Detection of immobilized nucleic acid on a membrane that has been released from lysed bacteria or phages.
5. In situ hybridization
Direct detection of nucleic acid in clinical specimens.
APPLICATIONS OF GENE PROBES
Gene probes have 3 basic applications in medicine:
1. Detection of specific nucleic acid sequences
Such sequences may be diagnostic of disease, e.g. the detection of a sequence unique to a particular
microorganism would demonstrate its presence in a specimen and, perhaps, confirm an infectious disease. This is
the principle of probes designed to detect and identify various infectious agents, including bacteria, protozoa and
viruses. Probes can be especially useful for detecting microorganisms that grow slowly (e.g. Mycobacterium
tuberculosis) or which cannot be cultured on artificial growth media (e.g. all viruses).
However, they are not usually capable of distinguishing between viable (live) and non-viable (dead) cells, which
is an important consideration with, for example, food poisoning organisms - many of which are not harmful
unless alive. Another problem is designing a probe to target a unique sequence so that it will only detect the
organism of interest. Sometimes an organism may show a unique biochemical characteristic and a probe can be
designed to target the gene of the enzyme involved. But it is rarely that easy!
2. Detection of changes to nucleic acid sequences
A change to the DNA sequence is a mutation, e.g. deletion, insertion, substitution. Changes in certain gene
sequences can cause inherited diseases and their detection by probes can be diagnostic. Unfortunately, with some
inherited diseases more than one type of mutation can cause the disease.
Some examples:
cystic fibrosis (due to a 3 bp deletion).
muscular dystrophies (due to various intragenic deletions).
phenylketonuria (due to various mutations).
apolipoprotein variants (some are due to a 1 bp mutation).
sickle cell anaemia (due to a 1 bp mutation).
a1-antitrypsin deficiency (due to approx. 50 different variants).
3. Detection of tandem repeat sequences
Tandem repeat sequences are 30-50 bps in length. Their size and distribution are distinctive for an individual.
They can be detected using probes and PCR. They are the basis of so-called "DNA fingerprinting" which was
developed by Alec Jeffreys at the University of Leicester, UK
It is used in forensic science to confirm the identity of a suspect from specimens left at the scene of a crime, e.g.
any body fluid, skin, hair.
This technique can also be used for paternity tests, sibling confirmation (or exclusion) and tissue typing.
