Radioactive Probes and Non-radioactive Probes
In molecular biology, nucleic acid hybridization is crucial, particularly for microbial diagnosis. It aids in the recognition or detection of a certain nucleic acid sequence. With the help of a probe, nucleic acids are anchored to a solid surface in this method. A DNA or RNA fragment that is complementary to an interesting sequence is known as a probe.
The probe will hybridise with the target sequence and make it identifiable if it is present in the sample. Probes can be classified as either radioactive or nonradioactive. As a result, we are able to tag the probes with fluorescent or radioactive tags. The target nucleic acid sequences are hybridised with the labelled probes, and the transcript with the greatest similarity is found using autoradiography and other imaging methods.
What is a Radioactive Probe?
Single-stranded DNA or RNA fragments with a radioactive tag are known as radioactive probes. Radioactive probes are made using radioisotopes. The radioactive elements 32P, 33P, and 35S are frequently employed to mark probes.
The labelling of probes also makes use, albeit to a lesser extent, of the radioisotopes 3H and 1251. They do, however, have distinct uses. When labelling radioactive probes, 32P is the radioisotope that is used the most frequently. Probes that use radioactivity offer a higher level of precision and accuracy.
As a result, they offer the highest level of sensitivity and enable precise measurement of target sequences. However, there are a number of drawbacks to radioactive probes. Their half-lives are brief. They are toxic and difficult to handle during production, usage, and disposal. Additionally, preparing radioactive probes is an expensive process.
What is a Non-Radioactive Probe?
The production class of chemically labelled probes is nonradioactive probes. Digoxigenin is an antibody-based marker and a nonradioactive probe. Digoxigenin probes have high sensitivity and specificity. Another label utilised in the creation of nonradioactive probes is biotin. The two nonradioactive probes that are most frequently employed in hybridization are Biotin/Streptavidin and Digoxigenin/Antibody-detection Systems.
Another nonradioactive probe technology is the horseradish peroxidase system. These nonradioactive probes can be spotted by autoradiography or other imaging methods after they have hybridised with the target sequences. In nucleic acid hybridization, nonradioactive probes are utilised more frequently than radioactive probes. This is so that nonradioactive probes are not connected to potentially dangerous substances.
Furthermore, the hybridization signal can be detected using nonradioactive detection techniques with shorter exposure durations. The procedures necessary for DNA hybridization with nonradioactive probes, however, are typically laborious and time-consuming. Furthermore, remedies that are sold commercially are pricey.
What Characteristics Do Radioactive and Nonradioactive Probes Share?
In nucleic acid hybridization, there are two types of probes: radioactive and nonradioactive.
They help the sample's target sequences to be found more easily.
Both kinds of probes are equally precise and sensitive.
What Distinguishes Radioactive Probes From Nonradioactive Probes?
Single-stranded DNA or RNA sequences labelled with radioactive isotopes are known as radioactive probes, whereas chemical tags are used to identify nonradioactive probes. The main distinction between radioactive and nonradioactive probes is thus this.
Additionally, radioactive isotopes present a risk. Therefore, radioactive probes pose a serious risk while nonradioactive probes do not. Additionally, the drawbacks of radioactive and nonradioactive probes are another distinction. The drawbacks of utilising radioactive probes include their brief half-lives and the dangers related to their manufacture, application, and disposal. However, the procedures needed to hybridise DNA using non-radioactive probes are typically laborious and time-consuming.
Practice Questions
1. Which of the following has no impact on how hybrid DNA develops?
Ionic strength
Pressure
Temperature
Homologous DNA
2. Describe a probe.
Chemically synthesised DNA
Purified DNA
Fragmented DNA duplex
Either purified or synthesised single-stranded DNA
Ans: The correct answer is option (d).
Conclusion
A nucleotide sequence that is complementary to the sequence of interest can be found in a segment of DNA or RNA known as a probe. The probes can be chemically, fluorescently, or radioactively tagged to detect the target sequence. In the sample, complementary sequences in the probes bind. While nonradioactive probes are marked with biotin, digoxigenin, or horseradish peroxidase, radioactive probes are marked with radioactive isotopes. The main distinction between radioactive and nonradioactive probes is thus this.
FAQs on Difference Between Radioactive and Nonradioactive Probes
1. Name the DNA which can be used as a probe.
A probe is a piece of radioactively labelled DNA that ranges in length from 100 to 1000 bases. To aid in the detection process, DNA probes are frequently marked with epitopes, fluorophores, radioisotopes, or biotin. The procedure of DNA fingerprinting involves the use of a DNA probe.
2. How are DNA probes being produced?
Recombinant DNA techniques can be used to create long DNA probes as plasmid inserts. Plasmid DNA linearization produces a DNA probe with a length of several hundred to a few thousand base pairs. To incorporate labels into this probe, a typical technique known as random priming or nick translation is performed.
3. State the reason behind Labelled RNA Probes?
By hybridising with a tagged RNA probe, certain nucleic acid sequences can be found. Sequences of varying lengths known as RNA probes are employed to find the presence of complementary nucleotide sequences in samples.
Gene probes are frequently marked with fluorescent or radioactive substances. The radioactive tag makes it possible to see how DNA binds. It could attach to a chromosome or a cell in the sample. A complementary sequence of a target nucleic acid interacts with a DNA or RNA fragment from the probe.