Spin 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 amine groups in tris).
Therefore, three stages are:
- The sample is added to the column and the nucleic acid binds due to the lower pH (relative to the silanol groups on the column) and salt concentration of the binding solution, which may contain 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 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 unicellular algae called diatoms which shield their cell walls with silica. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions. This was later improved used guanidinium thiocyanate or guanidinium hydrochloride as the chaotropic agent. The use of beads was later changed to minicolumns.
- For explanation of how the silica and DNA bind see separation by silica adsorption
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