What are Probes
Probes are something like that—searching a perfect matching, and bonding with each other. Matching sequences are selected for targeting a desirable sequence of the genome under investigation. The selected sequences are labeled with a particular detector that together searches their matching ones or we can say the complementary sequences. The complementary sequences have the tendency to bind each other, and the probed sequences show us the binding location or position. This fulfills our desirable investigation.
Definition- Probes are designed and labelled sequences, that helps in detection by binding specifically to the sequences of investigating genome, specifically the targeted sequences (DNA/RNA).
A probe can be synthesized artificially or by in-vitro methods of amplification.
Probes are mostly used in clinical diagnosis and detection of the microorganisms in various samples such as tissues, body fluids, excreta, etc.
Types of Probes:—
Basically, the Probes are of 2 types. DNA Probes and RNA Probes.
What are DNA Probes?
DNA probes are labeled single-stranded DNA sequences that are used to detect the complementary sequences in the provided sample. DNA probes are more convenient and preferable. Usually 100-1000 bp long DNA probes are used.DNA probes are synthesized mainly in end-labeled(basically labeled with the enzymes/chemicals at the 5’ end/3’ end of that probe’s sequence) or continuously labeled(randomly incorporated with enzymes/chemicals throughout the sequences) form by PCR amplification method. Labeled probes are used in hybridization techniques including- in-situ hybridization (ISH), Fluorescence in situ hybridization (FISH), plaque hybridization, DNA OR RNA hybridization to attach with the desired complementary sequences.
TaqMan probe, scorpion probe, and molecular beacons are the most abundantly used DNA probes.
What is TaqMan Probe?
TaqMan probe is the most popular, most significantly used probe in the biochemical world. This probe is made up of reporting dye (gives report by emission; can be fluorophore e.g.-carboxyfluorescein, tetrachlorofluorescein) at one end (5’) and quenching dye (it quenches the fluorescence e.g.-tetramethylrhodamine) at the other end (3’). Taqman recognizes the complementary site and binds to that site of DNA.
Normally reporting dye emits photon regularly but as TaqMan have a short length that means, two ends approximately close to each other, that’s why quenching dye quenches the emission, but after attachment of the probe in the desired segment, the Taq DNA polymerase attached with the reporting end along with primer and cleaves the reporting dye which then freely emits the photons and that emission is detected in the detector.
As elongation continuing by the polymerase, disattachment of TaqMan probe is held by 5’exonucleases activity and the probe is cleaved into segments. For a serious note Taq polymerase or any other thermostable enzyme, permits to perform well at a high temperature and of course, reduce the chances of enzyme-mediated point mutation. So everything depends upon the temperature where it should low & where it should be higher, that is the main funda. Meanwhile, we have to keep in mind about the temperature.
Molecular beacon is a more specific DNA probe. Generally, it is 20-25 nucleotide bases long. It has a loop segment(binds to the complementary sequence), stem segment(intra-pairing sequences), fluorophore end (5’), and quenching end (3’). Due to the very close proximity of the fluorophore end to the quenching end, the fluorophore cannot emit light. After recognition of the complementary sequences of the denatured DNA, the molecular beacon binds with it. The stem is found unfolds only when the loop segment is properly matched with the complementary sequences. The proper matching of loop segment bases and the complementary bases are more stable than the stem segment pairing. So, the unfolds stem segment leads to help the fluorophore end to go far apart from 3’ end and emits the lights which can be detected.
What are RNA Probes?
RNA probes are a fragment of RNA which detects the specific nucleic acids (especially mRNA in investigating biological sample) by hybridization. RNA probes are often known as riboprobes or cRNA probes. This probe is more reliable & has more strong thermodynamically stability when binds with target sequences as compared to the DNA probe.
RNA probe is synthesized by in-vitro transcription of the DNA template with the help of RNA polymerase at a definite binding site called promoters. Commercial plasmid vectors such as S6, T3, T7 with the promoters are cloned with the help of appropriate Restriction Endonuclease( which cuts at downstream of insert sequence & linearizes the plasmid template ), NTPs, RNase inhibitor( which inhibits the degradation of RNA), and RNA Polymerase( enhance the promoters synthesis reaction ).
RNA probes are got a very high degree of label incorporation as they are continuously labeled.
High sensitivity to degrade by RNase is a big disadvantage of RNA probes.
What are the Differences Between DNA and RNA Probes?
Labeling The Probes:
Labeling the probes is the main differential matter which can differentiate the probes from the DNA primers as their length are almost the same. Generally, either radio-isotopes (32P or 35S) or non-radio-isotopes (fluorophore) are used to label the probes. Though the autoradiography easily detects the labeled radioisotopes after hybridization easily, they are more difficult to handle & unsafe than non-radioactive molecules. So nowadays non-radioactive molecules are abundantly used such as fluorophore, biotin, digoxigenin, etc. However, their sensitivity is low. Nonradioactive labeling products are synthesized by definite translation where specific non-radioactive conjugated nucleotide bases are used. For example, biotin is synthesized by Nick-translation where biotin nucleotide bases are used. Traditionally, biotin could be incorporated through direct in-vitro transcriptional reactions via biotin-coupled rNTPs. While Digoxigenin is derived from the flowers and leaves of the Digitalis Purpurea plant. It’s mechanisms based on the Antibody sandwhich method & it also allows for more conjugated-antibody fragments to bind onto a single digoxigenin molecule and generates higher signal amplification.
Hybridization is the proper bonding with the probes and the complementary sequences of the target gene. Traditionally, it is done by Southern blotting (For DNA) or Northern blotting (for RNA). Basically in blotting, in the beginning, the targeted gene is fragmented by digestion of Restriction Endonuclease. In a nitrocellulose paper complementary radioactively labeled probes are allowed to bind with the targeted sequences if it is present. The unbound probes have to be washed out. The result is collected by autoradiography. On a serious note, the denaturing buffer has to be treated for denaturation of the ds DNA before treating the complementary probes.
Nowadays microarray technique is most significant for hybridization as it takes place more hybridization at once. Microarray is the small slice or glass slide which contains about 1000-100,000 parts. Each part contains known, SS DNA sequences in a particular pattern in a suitable environment. The complementary labeled probes are treated to hybridize if available in each part. Only fluorescent hybridized DNAs detect under the fluorescent microscope.
Moreover, In-Situ Hybridization (ISH), Fluorescent In-Situ Hybridization(FISH), Plaque Hybridization, DNA OR RNA Hybridization are also some Hybridization techniques.
Probes are used in –
- Identification and the quantification of the investigating nucleic acids.
- Disease diagnosis.
- Identification of microorganisms.
- Gene mapping.
- Forensic lab and gene profiling.
Nowadays it is very much useful for identifying hereditary syndrome and cancers.
Non-specific binding and sometimes weak interaction between probes and target sequences are the main issues for using the probes in molecular biology. In the case of high GC rich target sequences, sometimes probes are failed to bind properly.
As modern-era is totally interested and based on genetic research that means genetic identification, genetic model, genes responsible for various diseases, gene mapping, etc. From this point of view, Probes are the most important and significant agenda for this era.