2. Radioactive Probe
Introduction:
Radioactive probes are molecular tools that use radioactive isotopes to label specific molecules,
such as DNA, RNA, or proteins
These probes are particularly useful in molecular biology and genetic research for various
applications, including nucleic acid hybridization, gene expression studies, and protein
detection
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. But they are
used for specific applications.
3. 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.
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4. Here are some key points about radioactive probes:
1. Isotope Labeling:
Radioactive probes incorporate radioactive isotopes, typically P-32 (phosphorus-32) or S-35
(sulfur-35), into the target molecules
These isotopes emit radiation
2. Sensitivity:
Radioactive probes are highly sensitive and can detect even trace amounts of labeled
molecules
This sensitivity is useful for detecting low-abundance targets
5. 3. Safety Concerns:
Radioactive materials pose safety risks due to radiation exposure
Researchers working with radioisotopes must follow strict safety protocols and work in
specialized facilities
4. Environmental Considerations:
The disposal of radioactive waste is subject to strict regulations due to environmental concerns
Proper disposal and waste management are critical
5. Regulatory Oversight:
Researchers using radioactive materials are subject to regulatory oversight and must obtain
appropriate permits and comply with safety regulations
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6. Applications:
Radioactive probes have been historically used in a variety of molecular biology techniques:
Southern Blotting: Used to detect specific DNA sequences in a complex mixture
Northern Blotting: Similar to Southern blotting, but for the detection of specific RNA
sequences, providing insights into gene expression
Protein Labeling: Radioactive amino acids, like C-14 (carbon-14), can be used to label
proteins. This is often used in studies of protein turnover and metabolism
In Situ Hybridization (ISH): Used to detect and localize specific DNA or RNA sequences
within cells or tissues. It is valuable for studying gene expression patterns and identifying
chromosomal abnormalities
7. Non-radioactive probes
Introduction:
Non-radioactive probes are molecular tools used in
molecular biology and genetics to detect, identify,
and visualize specific DNA, RNA, or protein
sequences without the use of radioactive isotopes
These probes rely on various labeling methods, such
as fluorescent dyes, biotin, or enzymes, to tag the
target molecules
Non-radioactive probes offer safety, sensitivity, and
versatility and have become widely used in a range
of molecular biology techniques
8. Here are some common types of non-radioactive probes and their applications:
a) Fluorescent Probes:
Fluorescent in Situ Hybridization (FISH): Fluorescent probes are used to detect and
visualize specific DNA or RNA sequences in fixed cells or tissues
FISH is widely used in genetics and cytogenetics to identify chromosomal abnormalities
and gene mapping
b) Biotinylated Probes:
Biotin-Streptavidin Detection: Biotin-labeled probes can be used to tag DNA, RNA, or
proteins. Streptavidin, a protein that binds tightly to biotin, is often used to detect and
amplify signals
This method is utilized in techniques like Southern blotting, Northern blotting, and Western
blotting
9. c) Digoxigenin (DIG) Probes:
In Situ Hybridization (ISH): DIG-labeled probes are used to detect specific RNA sequences
in situ
They are detected with anti-DIG antibodies conjugated to enzymes, such as alkaline
phosphatase or horseradish peroxidase. ISH is valuable for gene expression studies
d) Enzyme-Labeled Probes:
Enzyme-Linked Immunosorbent Assay (ELISA): Enzyme-labeled probes are commonly
used in ELISA to quantify the presence of specific antigens (proteins) in a sample
Enzymes like horseradish peroxidase and alkaline phosphatase generate color or
luminescent signals
10. e) TaqMan Probes:
Quantitative Polymerase Chain Reaction (qPCR): TaqMan probes are used in real-time PCR to
quantify the amount of a specific DNA sequence
They contain a fluorescent reporter dye and a quencher, which are separated upon probe
cleavage during PCR amplification
f) DNA Microarray Probes:
Microarray Technology: Non-radioactive probes, typically fluorescently labeled, are used in
DNA microarrays to measure gene expression levels in a high-throughput manner
They allow for the simultaneous analysis of thousands of genes
g) Protein-Protein Interaction Probes:
Non-radioactive methods like the yeast two-hybrid system use non-radioactive probes to detect
and study protein-protein interactions, revealing potential protein binding partners
11. Advantages of Non-Radioactive Probes:
Safety: Non-radioactive probes eliminate the radiation hazards associated with
radioactive labelling
Versatility: They can be applied to a wide range of molecular biology techniques,
including genetic analysis, gene expression studies, and protein detection
Ease of use: These probes are often more convenient to handle and dispose of, reducing
safety concerns and regulatory compliance requirements
12. Applications:
Fluorescence in Situ Hybridization (FISH): Non-radioactive probes are widely used in FISH
to visualize and locate specific DNA sequences on chromosomes. FISH is used in genetics and
oncology for detecting chromosomal abnormalities and gene mapping
DNA Microarrays: Microarray technology utilizes non-radioactive probes to study gene
expression patterns by quantifying the levels of mRNA or DNA in a sample
Quantitative Polymerase Chain Reaction (qPCR): Non-radioactive probes, like TaqMan
probes, are used for real-time PCR to quantify the amount of specific DNA sequences in a
sample. This technique is essential for gene expression analysis and genetic testing
Next-Generation Sequencing (NGS): NGS libraries are often prepared using non-radioactive
probes to sequence DNA fragments, allowing for the identification of DNA variants, gene
expression, and more
