Diethyl pyrocarbonate

Diethyl Pyrocarbonate (DEPC) is a chemical compound with the formula (C2H5O)2CO2. It is a colorless liquid that is used primarily in molecular biology and biochemistry laboratories as a reagent for inactivating RNA-degrading enzymes known as ribonucleases (RNases). Due to its efficacy in this role, DEPC is a crucial tool in experiments involving RNA, such as transcription analysis and molecular cloning.

Properties

DEPC is highly reactive with amine groups, which makes it effective at modifying proteins and nucleic acids. This reactivity is the basis for its use in inactivating RNases, which are notoriously resistant to inactivation by heat and detergents. DEPC reacts with the histidine residues in RNases, thereby irreversibly inactivating the enzyme. However, DEPC is also hydrolyzed by water, with a half-life of approximately 30 minutes at pH 7 and 25°C, resulting in the production of carbon dioxide and ethanol. This hydrolysis reaction is accelerated in the presence of buffers and at higher temperatures.

Usage

Before working with RNA, laboratory equipment and solutions are often treated with DEPC to inactivate RNases. Typically, a 0.1% solution of DEPC in water or buffer is prepared, and the items to be decontaminated are immersed in this solution overnight, followed by autoclaving to remove any residual DEPC. It is crucial to ensure that DEPC is completely removed after treatment since it can modify nucleic acids and interfere with subsequent experiments.

Safety

DEPC is considered a hazardous substance and must be handled with care. It is a potential carcinogen and can cause irritation to the skin, eyes, and respiratory system. Appropriate safety measures, including the use of gloves, goggles, and fume hoods, are essential when working with DEPC.

Applications in Molecular Biology

DEPC's primary application is in the preparation of RNase-free solutions and equipment for RNA work. Its ability to inactivate RNases without leaving residues that interfere with RNA integrity or function makes it invaluable in molecular biology. DEPC treatment is a standard step in protocols for RNA isolation, cDNA synthesis, and in vitro transcription.

Limitations

While DEPC is effective at inactivating RNases, it cannot be used with Tris-based buffers or solutions containing primary amines, as it reacts with these compounds. Additionally, DEPC cannot be used to treat certain materials, such as polycarbonate, because it can cause degradation.

Alternatives

For applications where DEPC treatment is not suitable, other methods for inactivating RNases, such as UV irradiation or the use of RNase inhibitors, may be employed. These alternatives, however, may not be as effective or may introduce other limitations.