Radioactive or non radioactive probes
What are Radioactive Probes?
Radioactive probes are the single-stranded DNA or RNA fragments with a radioactive tag.
Radioisotopes are used in preparing radioactive probes. Radioisotopes 32P, 33P and 35S are
commonly used in the labeling of probes. Moreover, radioisotopes 3H and 1251 are also used to
a lesser extent in the labeling of probes. Among different radioisotopes, 32P is the most
commonly used isotope in labelling radioactive probes.
Radioactive probes provide a higher degree of reliability and specificity. Therefore, they
provide maximum sensitivity and allow accurate quantification of target sequences. However,
there are several disadvantages associated with radioactive probes. They have short half-lives.
Moreover, they are hazardous and production, use and disposal are problematic when
handling. In addition, radioactive probe preparation is a costly process. Therefore, due to the
safety issues and cost, radioactive probes are not used as nonradioactive probes nowadays.
What are Nonradioactive Probes?
Nonradioactive probes are the second type of probes that are chemically labelled.
Digoxigenin is a nonradioactive probe, which is an antibody-based marker. Digoxigenin
probes are specific and sensitive. Biotin is another label used in nonradioactive probe
preparation. Biotin/Streptavidin and Digoxigenin/Antibody-detection systems are the most
commonly used nonradioactive probes in hybridization. Furthermore, horseradish peroxidase
system is another nonradioactive probe system. Once these nonradioactive probes are
hybridized with the target sequences, they can be detected via autoradiography or other
imaging techniques.
Nonradioactive probes are used more often in nucleic acid hybridization than radioactive
probes. This is because nonradioactive probes are not associated with hazardous materials.
Furthermore, nonradioactive detection methods require shorter exposure times to detect the
hybridization signal. However, the steps involved in DNA hybridization with nonradioactive
probes are usually tedious and time-consuming.
What are the Similarities Between Radioactive and Nonradioactive Probes?
Radioactive and nonradioactive probes are two types of probes used in nucleic acid
hybridization.
They facilitate the detection of target sequences in the sample.
Both types of probes are equally sensitive and specific.
Difference between Radioactive and non radioactive isotopes or probes
Different types of non radioactive isotopic procedure and their application
Biotinylated probes bind to streptavidin conjugated to an enzyme or fluorophore.
Applications:
o Detection of DNA/RNA hybrids in blots.
o ELISA for protein detection.
o Pull-down assays in proteomics.
Advantages:
o High specificity due to strong biotin-streptavidin affinity.
o Flexibility in detection methods (colorimetric, chemiluminescent, or
fluorescent).
e) Chemiluminescent Detection
Principle: Chemical reactions produce light as a byproduct, which is detected using
luminometers.
Applications:
o Western blotting with enzyme-labeled antibodies (e.g., HRP or AP).
o Southern or Northern blotting for DNA/RNA detection.
o ELISA for protein quantification.
Advantages:
o High sensitivity and signal stability.
o No requirement for hazardous radioisotopes.
f) Colorimetric Methods
Principle: Enzyme reactions produce color changes measurable with
spectrophotometers.
Applications:
o Nucleic acid quantification using dyes like Bicinchoninic Acid (BCA) or
Bradford reagent for protein.
o Dot blot assays with enzyme-conjugated probes.
Advantages:
o Simple and inexpensive.
o Visual readout without special equipment.
g) Mass Spectrometry
Principle: Analytes are ionized, separated by their mass-to-charge ratio, and detected.
Applications:
o Protein identification and quantification in proteomics.
o Metabolomics and post-translational modification analysis.
Advantages:
o High precision and throughput.
o Non-invasive and non-radioactive.
h) Real-Time PCR (qPCR)
Principle: Monitors amplification of DNA using fluorescent dyes or probes (e.g.,
SYBR Green, TaqMan).
Applications:
o Quantitative gene expression studies.
o Pathogen detection.
o SNP genotyping.
Advantages:
o High sensitivity and specificity.
o Real-time monitoring of amplification.
i) Surface Plasmon Resonance (SPR)
Principle: Measures molecular interactions in real-time using changes in refractive
index.
Applications:
o Protein-protein and protein-DNA binding studies.
o Small molecule screening for drug targets.
Advantages:
o Non-invasive, no labeling required.
o Quantitative binding affinity analysis.
