Column-based nucleic acid purification
- Column-based nucleic acid purification
Column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids.
This method relies on the fact that the nucleic acid may bind (adsorption) to the solid phase (silica or other) depending on the pH and the salt content of the buffer, which may be a Tris-EDTA (TE) buffer or Phosphate buffer (used in DNA microarray experiments due to the reactive amines).
Therefore, three stages are:
*The sample is added to the column and the nucleic acid binds thanks to the high pH and salt concentration of the binding solution, which may contain other than the buffer, a denaturing agent (such as guanidine hydrochloride),Triton X-100, isopropanol and a pH indicator
*The column is then washed (5 mM KPO4 pH 8.0 or similar, 80% EtOH)
*The column can be eluted with buffer water or simply water
Even prior to the major techniques employed today it was known that in the presence of chaotropic agents, such as Sodium iodide or Sodium perchlorate, DNA binds to silica, glass particles or to diatoms, unicellular algae called who shield their cell walls with silica . This propriety was used to purify nucleic acid using glass powder or silica beads under alkaline conditions. [Marko MA, Chipperfield R, Birnboim HC. A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem. 1982 Apr;121(2):382-7.PMID: 6179438] This was later improved used guanidinium thiocyanate or guanidinium hydrochloride as the chaotropic agent. [Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495-503. PMID: 1691208] The use of beads was later changed to minicolumns and currently the main manufacturer is Qiagen and as a result they are often referred to as Qiagen columns.
:"For explanation of how the silicon and DNA bind see separation by silica adsorption
ee also
*Guanidinium thiocyanate-phenol-chloroform extraction
*Ethanol precipitation
*DNA separation by silica adsorption
References
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