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. their complementarity 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.
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):
Amplification of target sequence
To increase the sensitivity of a probe system the target sequence can be amplified (i.e. more copies made of it). This enables detection of very small amounts of the target which may be undetectable without amplification.
Target DNA can be amplified using the polymerase chain reaction (PCR) or using ordinary DNA polymerase. Both methods use primers but PCR produces exponential amplification of the target whereas the second method produces arithmetical amplification and less target in a given time.
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.
Both these techniques are covered in Section 4 (Analysis of DNA).
This term can be defined as:
"The conditions of hybridization that increase the specificity of binding between two single-stranded portions of nucleic acid."
(Stringency can be applied to any hybridization reaction, not just probe and target, but it is convenient to cover it here. The same principles apply, for example, to hybridization of a primer to a sequence prior to DNA replication.)
The degree and specificity of binding between two portions of nucleic acid depends on hydrogen bonding between bases on opposite strands and is affected by 4 external factors:
1. Temperature
2. pH
3. Salt concentration
4. Presence of denaturants
Nucleic acid hybrids can tolerate a certain proportion of mismatched base pairs (mismatches) and still form stable duplexes, i.e. remain double-stranded.
Normal pairings in DNA are A=T and G=C (with double and triple hydrogen bonds respectively).
Mismatches are: A/G, A/C, G/T and C/T which do not form hydrogen bonds.
The greater the proportion of mismatches, the greater the chances of the two molecules separating (denaturing). However, under conditions of low stringency two nucleic acid strands which are, perhaps, only 80% homologous may still remain together. Under conditions of high stringency the same two strands would separate. Therefore, altering the conditions of stringency can be used to assess the degree of homology or complementarity between two nucleic acid strands.
Stringency can also be regarded as measures of the degree of mismatch that can be tolerated, and the level of specificity of a probe (or primer) for its target. This is summarized in the table below:
| LOW STRINGENCY | HIGH STRINGENCY | |
TEMPERATURE |
LOW |
HIGH |
pH |
NEUTRAL |
EXTREME |
SALT CONCENTRATION |
HIGH |
LOW/ZERO |
DENATURANT CONCENTRATION |
LOW/ZERO |
HIGH |
TOLERANCE OF MISMATCHES |
HIGH |
LOW |
SPECIFICITY |
LOW |
HIGH |
Of the 4 external factors affecting hybridization, temperature is the easiest to control and use to assess the specificity of a probe for its target.
High stringency is not always required as it increases specificity of probe for its target. If probe is not a perfect match for the target (perhaps because exact sequence of target is not known or because the target varies in different samples) it would be best to lower the conditions of stringency to ensure hybridization.
Also, by varying stringency, it is possible to use one probe for different tasks, e.g. a probe for Campylobacter bacteria will hybridize and identify the species Campylobacter jejuni only when used under high stringency , but under low stringency will identify any Campylobacter species. In this case, stringency can be altered depending on what bacteria require identification.
Mid-point temperature (Tm)
Tm is the temperature at which half (50%) of the hybridized nucleic acid molecules in a sample denature, i.e. strand separation occurs. This is used because in a population of nucleic acid molecules there will always be some variation and also because of chance they do not all denature simultaneously at a particular temperature. Measuring the Tm gives an indication of the complementarity between probe and target (i.e. how closely their base sequences match). Tm also provides a measure of the specificity of a probe for its target - the higher the Tm the greater the specificity.
The graph below illustrates the measurement of the mid-point temperature from a graph of percentage hybridization against temperature. The temperature axis has no values because these will vary depending on the degree of complementarity between probe and target. If complementarity is high the graph will be shifted to the right and Tm will be high. If complementarity is low, the graph will be shifted to the left and Tm will be lower.
The slope of the graph provides an indication of the homogeneity of the population of hybridizing nucleic acids molecules, i.e. their uniformity. The steeper the graph, the more homogeneous the population.
SHORT v. LONG PROBES
Short, oligonucleotide probes may be only 14-40 bases in length. Long probes may be 100's of bases in length. Under conditions of high stringency, and other optimal conditions, a probe is capable of detecting a one base pair (bp) change in nucleic acid.
Advantages of short probes (compared to long):
more stable with time 
longer shelf life.
simpler to make.
hybridize at more rapid rates, e.g. short probes may have reaction time of less than 30 minutes. (C.F. long probes may have reaction time of 4-16 hours.)
Disadvantages (compared to long):
the amount of label that each probe molecule can carry is more limited by its shorter length.
sensitivity may be less (for above reason).
The above problems can be overcome to some extent by: amplifying the target, thus allowing more probe molecules to hybridize and so increasing the signal and sensitivity; and/or amplifying the signal from the probe label (reporter molecule).
Long probes are more tolerant of mismatches than short probes but it is the proportion of mismatches that counts not the actual number. Therefore, a short probe with 2 mismatches is less likely to hybridize to a target than a long probe with 2 mismatches. Under standard conditions of use it would be expected that short probes would be more specific for their target than long ones.
USES OF GENE PROBES
Probes hybridize to complementary DNA or RNA sequences and have several uses:
Detection of gel-fractionated DNA molecules transferred to a membrane. This includes restriction fragment length polymorphism (RFLP) analysis.
As above but used for RNA.
3. Dot blots
Detection of unfractionated nucleic acid immobilized on a membrane.
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!
There is more discussion of nucleic-acid based methods for the detection and identification of microorganisms in the PCR section. Click to view.
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. In which case, a probe may have to be used under low stringency (to allow hybridization to a range of sequences) or several probes used (a "battery") to ensure that the target is "hit".
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.
Q. Work out who is the criminal (or father!) from the following gel prints:
You could look up more details on this technique in one of the suggested references.
END
OF SECTION 7.
NOW GO TO SECTION 8
(DNA AMPLIFICATION).
This is the commonest method of labelling DNA probes but the term "nick translation" is confusing. It has nothing to do with the translation of mRNA into polypeptide chains.
Stages:
1. Nicks are introduced into a DNA duplex using DNAse I enzyme which produces random breaks in the strands.
2. This damage is then repaired by the addition of DNA polymerase I enzyme in the presence of free labelled nucleotides.
3. Labelled nucleotides are incorporated into DNA molecule which then becomes labelled.
More label can be incorporated into the probe by allowing the reaction to proceed for a longer time.
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Stages:
1. DNA of interest is denatured to give single-strands.
2. Random primer sequences are added (or sequences unique to a particular sequence of interest).
3. DNA polymerase is added together with labelled nucleotides.
4. Complementary DNA strands are synthesized starting from the primer sequences and incorporating the labelled nucleotides. There is partial filling in of the gaps between the primers.
5. DNA is then denatured to release the labelled probe molecules.
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RNA polymerase enzyme and labelled ribonucleotides are used to synthesized RNA probes using DNA as a template.
DNA template + RNA polymerase + labelled ribonucleotides
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Label is attached to either 3' or 5' end of linear DNA or RNA.
e.g. T4 polynucleotide kinase can be used to catalyze the transfer of labelled phosphate from a nucleoside donor to the 5' - hydroxyl group of a polynucleotide, oligonucleotide or nucleoside.
Activity (and hence sensitivity) is generally less than with other labelling methods because the amount of label is limited by the number of ends!!!
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Nucleic acids can be labelled directly by the addition of labelled materials that combine with them. The process is equivalent to "staining".
e.g. iodination of DNA using 125I.
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