Скачать или смотреть what is Nucleic acid probe | hybridization probe | radioactive and non - radioactive probe

what is Nucleic acid probe | hybridization probe | radioactive and non - radioactive probe


In this video following topics explain :-

1. what is Nucleic acid probe / hybridization probe (DNA and RNA probes)

A nucleic acid probe is a single-stranded sequence of nucleic acids (either DNA or RNA) that is used to detect the presence of complementary sequences of nucleic acids in a sample. These probes are labeled with a radioactive or fluorescent tag, allowing them to be easily detected when they hybridize or bind to their complementary target sequence.

2. Non - radioactive probe

Non-radioactive probes are nucleic acid probes that are labeled with non-radioactive markers, making them safer and often more convenient to use compared to traditional radioactive probes. These probes use chemical labels that can be detected through various methods, such as fluorescence, chemiluminescence, or colorimetric detection.

*Types of Non-Radioactive Labels:*

1. **Fluorescent Labels**:
**Fluorescein**: A common fluorescent dye that emits green light when excited by blue light.
**Rhodamine**: Another fluorescent dye, which emits red light when excited.
**Cy3, Cy5**: Fluorescent dyes used in more advanced techniques, such as microarray analysis and FISH.

2. **Enzyme Labels**:
**Alkaline Phosphatase (AP)**: An enzyme that catalyzes a reaction to produce a color change or light emission, depending on the substrate used. Detection is usually performed using chemiluminescent or colorimetric substrates.
**Horseradish Peroxidase (HRP)**: Another enzyme commonly used, which can produce a detectable signal through a chemiluminescent or colorimetric reaction.

3. **Biotin-Avidin/Neutravidin System**:
Probes labeled with biotin (a small molecule) can be detected using avidin or streptavidin, proteins that bind strongly to biotin. These proteins can be conjugated to an enzyme (like HRP or AP) or a fluorescent molecule, allowing for signal detection.

4. **Digoxigenin (DIG)**:
A plant-derived steroid used as a label, detected by anti-digoxigenin antibodies conjugated to an enzyme like AP or HRP. This is a highly specific and sensitive method.


3. Radioactive probe

A radioactive probe is a nucleic acid probe labeled with a radioactive isotope, used to detect specific DNA or RNA sequences in molecular biology experiments. These probes have been a staple in techniques like Southern blotting, Northern blotting, and in situ hybridization due to their high sensitivity.

*Key Aspects of Radioactive Probes:*

1. **Labeling**:
Radioactive probes are tagged with isotopes such as:
**Phosphorus-32 (³²P)**: A common choice due to its high-energy beta emissions, providing strong signals but with a relatively short half-life of 14.3 days.
**Sulfur-35 (³⁵S)**: Used in RNA labeling; it emits lower-energy beta particles with a longer half-life of 87.4 days.
**Tritium (³H)**: Has a very long half-life (12.3 years) and emits low-energy beta particles, useful for high-resolution studies but with weaker signals.

2. **Detection**:
**Autoradiography**: The most common detection method, where the probe bound to its target is exposed to photographic film or a phosphor screen. The radioactive emissions create an image on the film, revealing the location of the hybridized probe.
**Scintillation Counting**: Measures the radiation emitted from the probe in liquid samples, often used in quantitative studies.

3. **Applications**:
**Southern Blotting**: For detecting specific DNA sequences within a mixture of DNA fragments separated by gel electrophoresis.
**Northern Blotting**: Similar to Southern blotting but for RNA detection.
**In Situ Hybridization (ISH)**: Detecting specific nucleic acids in fixed tissues or cells.
**DNA Footprinting**: Identifying the DNA regions protected by protein binding.

4. **Advantages**:
**High Sensitivity**: Radioactive probes can detect very low amounts of nucleic acids, making them particularly useful in cases where the target is scarce.
**Quantitative**: The intensity of the signal can be measured to quantify the amount of target nucleic acid.

5. **Disadvantages**:
**Safety Risks**: Handling radioactive materials requires strict safety protocols to protect users from exposure.
**Short Shelf-Life**: The decay of radioactive isotopes reduces the probe’s effectiveness over time, necessitating careful timing in experiments.
**Disposal Challenges**: Radioactive waste must be disposed of according to stringent regulations, which can be costly and complex.

**Current Use**:
Though highly effective, the use of radioactive probes has decreased in favor of non-radioactive alternatives due to safety and environmental concerns. However, they are still employed in some specialized applications where their unmatched sensitivity is necessary.