“DNA probes are the known short, single-stranded, labelled DNA sequences used to detect the presence or absence of nucleic acid in a sample.”
In situ hybridization allows the use of the DNA or RNA probes to employ in the detection of various nucleic acid present in any biological sample.
Therefore it is used in medical industries, food industries, microbial identification and environmental science.
In general, the nucleic acid probes are 100 to 10000 basepair long, tagged with the coloured molecule either radioisotopes or fluorochromes.
The aim of using the DNA probe is to identify the presence of a gene or DNA sequence of interest present on a chromosome or in a sample.
A probe can be synthesised artificially or by the in vitro methods or amplification methods.
Type of probe:
Based on our sample, the nucleic acid hybridization probe might be a DNA probe or RNA probe:
The DNA probe is used to detect the presence of the DNA or a gene present in a sample. 100 to 10000bp long complementary DNA sequence is used to detect the presence of complementary DNA sequence.
For that, the single-stranded probe is first prepared by denaturing it and employed it for hybridization on the nitrocellulose paper.
The DNA probe can also be synthesised chemically or prepared using the amplification method or using the cDNA library.
The DNA probes are adopted for detection of the microorganism present in a sample and DNA quantification.
DNA probes are the first choice in the FISH- fluorescence in situ hybridization in which probes are allowed to hybridize directly on the chromosome.
The computational software and high-end microscopes used to detect the results.
TaqMan probe, scorpion probe and molecular beacons are commonly used DNA probes in the realtime PCR.
Examples of DNA probe:
The TaqMan probe is one of the most popular types of probe chemistry available nowadays.
A type of hydrolysis probe, commonly used in the real-time PCR assay, and developed by Kary Mullis, himself- the founder of PCR.
The chemistry of the TaqMan probe is based on the exonuclease activity of the Taq DNA polymerase enzyme.
The one end (5’) of the probe contains a fluorophore while the other end (3’) contains the quencher molecule which is in close proximity of the fluorophore.
Once the probe finds its complementary sequence, it binds to it, the DNA polymerase starts synthesising the strand, but removes the probe by the 3’ to 5’ exonuclease activity.
The probe unquenched, the fluorophores detached from the quencher molecule and emits the fluorescent.
The probe is hydrolysed and the fluorescent emitted by it is measured by the detector. This is the principle of the TaqMan probe.
Taqman probes are widely used in the DNA or gene quantification and gene expression studies. Some of the commercially available probes avail researchers for rapid detection and quantification of microorganism.
For increasing the specificity of the reaction and results, the molecular beacons like hairpin probes are used in the quantification assay.
Structurally, the molecular beacons contain two complementary ends attached with each other with a fluorophore at one end and a quencher molecule at another end. The quencher dye is in close proximity with the fluorescent molecule. Due to this, the fluorophore can not emit fluorescence.
Once the probe finds its complementary sequence, it binds to it and the fluorophore unquenched- emits fluorescence. We have covered an entire article on it, read it here: Molecular beacons.
RNA probes are often known as riboprobes or cRNA probes.
It is used to detect the presence of mRNA present in the biological sample thereby detection of gene expression.
Traditionally it is used in the northern blot hybridization method in which once the RNA probes are constructed, it is employed for hybridization on the nitrocellulose paper.
On the nitrocellulose paper, our target single-stranded nucleic acid already immobilised, by doing the autoradiography, the hybridization signal can be detected.
In the recent day, the RNA probes are used in the microarray in which millions of probes are immobilised on the solid surface.
Employing the nucleic acid allows hybridization of only complementary DNA sequence and therefore many different copy number variations can be detected in the single assays.
Although, the RNA probes are only used for studying the gene expression.
Labelling of probe:
One of the important characteristics of the probe is the label attached on its end which makes it different from the DNA primers.
Either radioactive molecule or non- radioactive molecule are used to label the probe. Radio-active molecules such as 32P or 35S are used to do so.
Autoradiography is used to detect the hybridization, however, the radiolabelled molecules are not safer to use.
In order to achieve safety, non-radioactive compounds, such fluorophores are now used to label the probe which is safer than the radioactive compounds, however, the sensitivity is low.
Other molecules such as biotin and digoxigenin are also used in the labelling of the probe as well.
How probe hybridization occurs?
Traditionally, the method is known as southern blot (for DNA hybridization) or northern blot (for RNA hybridization).
In the very first step, the DNA sequence of our interest is digested with the restriction endonuclease and allow to immobilised on the nitrocellulose paper.
A complementary probe is selected, labelled and denatured first.
Then in the next step, the probe mixture allows binding on the nitrocellulose paper, if it finds the exact complementary sequence, it will bind.
The unbound probes are removed by washing the nitrocellulose paper with the wash buffer.
The final results are collected using the autoradiography method.
In the modern version of this technique- called a microarray, the probes are hybridized instead of sample DNA.
The sample is allowed to hybridize on the surface containing millions of probe, thus microarray enables to screen many mutations or alterations at once.
Quantification of nucleic acid- one of the important application of the probe-based method is in the quantification of nucleic acid- DNA or RNA present in the sample.
Besides this, it is used in the microbial identification/quantification, disease diagnosis and gene mapping.
It is also used in forensic science and DNA profiling studies.
Non-specific binding is one of the key limitations of using DNA probes in molecular genetics.
Sometimes the probe binds to the less complementary sequences and emits the fluorescence, also in some higher GC rich template the probe can not be bind properly.
In both cases, positive results can not be obtained.
DNA probes are one of the important elements of the molecular genetic studies since long and we can say, it is successfully used till now. Different types of DNA probes are now commercially available.
However, one can construct the DNA probe in-house but the process is inconsistent, tedious and time-consuming.