DNA and RNA extraction

DNA, RNA, and protein extraction is a basic method used in molecular biology. There are now specialized methods of extraction, namely Solution-based extraction and Column-based extraction protocols, instead of more time-consuming and limited processes.

Purification of DNA, RNA, and proteins requires the disruption of cells or tissue, the denaturation (or unbinding or nucleaoprotein chains), and inactivation of nucleases. Contaminants such as carbohydrates and lipids should be removed as well.

Extraction Methods (with Chemical Use)

 * The Guanidinium thiocyanate - Phenol - Chloroform extraction method is commonly used. Phenol denatures proteins rapidly, but does not completely inhibit RNA activity. When phenol and chloroform are used in conjunction, a biphasic emulsion forms. A hydrophobic layer of the emulsion settles on the bottom, and a hydrophilic layer on the top. The upper layer contains DNA, and can be extracted by the adding ethanol and salt. Guanidinium thiocyanate is used in protein degredation.


 * The Alkaline Extraction method isolates DNA and E.coli. Covalently Closed DNA does not denature in the prescence of Sodium Dodecyl Sulfate, while chromosonal DNA denatures and is coated with the SDS.


 * The CTAB Extraction Method (Cetyltrimethylammonium bromide) first requires material to be ground to break cell walls. CTAB is a nonionic detergent, and can therefore precipitate nucleic acids from low ionic strenghth solutions. Proteins and neutral polysaccharides remain in solution. CTAB only works under low ionic conditions, so CTAB is only useful when the nucleic acid is from organisms with high levels of polysaccharides (plants and Gram-negative bacteria).


 * Ethidium Bromide is used in conjunction with Cesium Chloride in a centrifuge. This method is complicated and time consuming and requires a large bacterial culutre. The extraction is density based, and Covalently Closed circular molecules will gather at low densities (there is less Ethidium Bromide) and can be precipitated with alcohol. The Ethidium Bromide is removed by a hydrophobic solvent.

source: Tan, Siun Chee, and Beow Chin Yiap. "DNA, RNA, and Protein Extraction: The Past and Present." J. Biomed Biotechnol. 30 November 2009. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789530/

Alternatives

 * Silica Matrices: This process uses glass particles and hydrated silica matrix (prepared in sodium hydroxide or potassium hydroxide). DNA binds to this inorganic matrix and is released in heated water. This method works through the affinity of the negative DNA charge to the positively charged silica particles. Sodium is a cation under high salt conditions, breaking hydrogen bonds in water and silica.
 * Diatomaceous Earth, or diatomite has a silica content of 94%. This process binds DNA to the particles.
 * Magnetic Bead Based Nucleic Acid Purification: this is simple and efficient.

Tan, Siun Chee, and Beow Chin Yiap. "DNA, RNA, and Protein Extraction: The Past and Present." J. Biomed Biotechnol. 30 November 2009. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789530/