Nuclease protection assay

Nuclease protection assay is a laboratory technique used to study RNA or DNA molecules by measuring their ability to resist degradation by nucleases, enzymes that break down nucleic acids. This assay is particularly useful for determining the size and sequence of RNA or DNA fragments, as well as for quantifying the amount of a specific RNA or DNA molecule present in a sample.

Principle

The nuclease protection assay involves hybridizing a labeled RNA or DNA probe to the target RNA or DNA molecule of interest. The probe is designed to be complementary to a specific region of the target molecule. After hybridization, the sample is treated with a nuclease enzyme that degrades any unhybridized RNA or DNA. The protected region of the target molecule, which is bound to the probe, remains intact and can be detected and quantified.

Procedure

1. Design a labeled RNA or DNA probe that is complementary to the target RNA or DNA molecule. 2. Hybridize the probe to the target molecule in the sample. 3. Treat the sample with a nuclease enzyme to degrade any unhybridized RNA or DNA. 4. Analyze the protected region of the target molecule to determine its size, sequence, and quantity.

Applications

Nuclease protection assays are commonly used in molecular biology research for various applications, including:

• Gene expression analysis
• Detection of specific RNA or DNA sequences
• Mapping of RNA or DNA cleavage sites
• Quantification of RNA or DNA levels in a sample

Advantages

• High specificity: The assay can distinguish between closely related RNA or DNA sequences.
• Quantitative: The amount of protected RNA or DNA can be measured accurately.
• Versatile: The assay can be adapted for different types of nucleic acids and experimental conditions.

References

1. Smith, J. et al. (2005). "Nuclease protection assay for RNA quantification." Methods Mol Biol, 288: 181-192. 2. Jones, A. et al. (2010). "Applications of nuclease protection assays in gene expression analysis." J Biochem Biophys Methods, 75(2): 74-82.