Explore the process of DNA extraction in microbiology, including methods, stages, chemicals used, and various techniques such as organic and physical extraction. Learn about the steps involved in isolating DNA from biological samples and the importance of purification for obtaining pure DNA samples ready for analysis.
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Definition of DNA extraction DNA extraction: is a process that used to purify DNA by using chemical or physical methods from a biological sample separating DNA from protein, cell membranes, and other cellular components. Or Isolating DNA by disrupting cell wall/cell membrane and a nuclear membrane is called a DNA extraction . DNA extraction protocol involves: 1.lysis of membranes for DNA extraction. 2. separating DNA from impurities like proteins, and other substances.
General Stages of DNA Extraction are: Cell dissolution: lysis of cell and the nucleus to extract DNA into the buffer. Precipitation: removing the impurities and proteins from the sample. Purification: This final stage is done to get a completely pure DNA sample ready to be used. Note: the main steps remain similar among all DNA extraction methods.
Steps in DNA extraction: Steps in DNA extraction: Cell membrane or cell wall lysis Nuclear envelope lysis Removal of contaminant Stabilizing DNA DNA Precipitation DNA washing and elution
Chemical-based DNA extraction method Organic DNA extraction Phenol-chloroform and isoamyl alcohol Inorganic DNA extraction Proteinase K DNA extraction CTAB DNA extraction SDS DNA extraction Salting out method Silica-gel-based techniques Physical DNA extraction methods Magnetic bead DNA extraction Paper DNA extraction
The solution-based or chemical-based DNA extraction method is subdivided into: Organic solvent-based DNA extraction This method depends on the use of organic substances such as phenol and chloroform. Example: Phenol-chloroform and isoamyl alcohol Inorganic solvent-based DNA extraction It depends on the inorganic solvents. Example: Proteinase K DNA extraction, Salting out method, SDS DNA extraction CTAB DNA extraction, Silica-gel-based techniques
Liquid-liquid DNA extraction: One of the commonest methods for nucleic acid extraction is liquid-liquid extraction. Here only solutions prepared by various chemical compositions are used for extraction. The overall success of the experiment depends on how effectively the lysis buffer is prepared. Lysis buffer is often prepared in one or two solutions as it uses many chemicals. Common chemicals for liquid-liquid DNA extraction are Phenol, chloroform, isoamyl alcohol, SDS, CTAB, Triton X100, Tris, EDTA, MgCl2, NaCl, PVP, beta- mercaptoethanol and other detergents. These methods commonly require centrifugation for separation. Common liquid-liquid DNA extraction methods are, Phenol, chloroform and isoamyl alcohol DNA extraction CTAB DNA extraction SDS DNA extraction
Solid-liquid phase DNA extraction: Spin-column and magnetic bead-based separation techniques are solid- liquid phase DNA extraction that uses both liquid lysis buffer and solid phases like silica gel or magnetic beads. These groups of techniques are safe, fast and easy to perform but at the same time are costly.
DNA extraction by chromatography: Type: Physical separation technique Subtype: Solvent-based DNA extraction Chromatography-based nucleic acid separation, however, is not used in recent times but was part of the history of DNA extraction. SEC (Size exclusion chromatography) and IEC (Ion exchange chromatography) were certainly popular for separation.
