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Almost all the experiments dealing with gene manipulations require pure forms of either DNA or RNA, or sometimes even both. Hence there is a need for the reliable isolation of nucleic acids from the cells. The purification of nucleic acids broadly involves three stages.

1. Breaking or opening of the cells to expose nucleic acids.
2. Separation of nucleic acids from other cellular components.
3. Recovery of nucleic acids in a pure form.

Analytical procedures involving a few steps to several steps are in use for the purification of nucleic acids. In fact, commercial kits are readily available these days to enable purification of either DNA or RNA from different sources.

The basic principle and procedures for nucleic acid purification

Purification of cellular DNA

The first step for DNA purification is to open the cells and release DNA. The method should be gentle to preserve the native DNA. Due to variability in cell structure, the approaches to break the cells are so different.

Methods to purify DNA

There are two different approaches to purify DNA from the cellular extracts.

Purification of DNA by removing cellular components: This involves the degradation or complete removal of all the cellular components other than DNA. This approach is suitable if the cells do not contain large quantities of lipids and carbohydrates. The cellular extract is centrifuged at a low speed to remove the debris that forms a pellet at the bottom of the tube. The supernatant is collected and treated with phenol to precipitate proteins at the interface between the organic and aqueous layers. The aqueous layer, containing the dissolved nucleic acids, is collected and treated with the enzyme ribonuclease. The RNA is degraded while the DNA remains intact. This DNA can be precipitated by adding ethanol and isolated after centrifugation, and suspended in an appropriate buffer.

Direct purification of DNA: In this approach, the DNA itself is selectively removed from the cellular extract and isolated. There are two ways for direct purification of DNA.

In one method, the addition of a detergent cetyltrimethyl ammonium results in the formation of an insoluble complex with nucleic acids. This complex, in the form of precipitate is collected after centrifugation and suspended in a high-salt solution to release nucleic acids. By treatment with Rnase, RNA is degraded. Pure DNA can be isolated by ethanol precipitation.
The second technique is based on the principle of tight binding between DNA and silica particles in the presence of a denaturing agent such as guanidinium thiocyanate. The isolation of DNA can be achieved by the direct addition of silica particles and guanidinium thiocyanate to the cellular extract, followed by centrifugation. Alternately, a column chromatography containing silica can be used, and through this the extract and guanidinium thiocyanate are passed. The DNA binds to the silica particles in the column, which can be recovered.