Spin Column-based Nucleic Acid Purification
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Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify
nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main ...
s. This method relies on the fact that nucleic acid will
bind BIND () is a suite of software for interacting with the Domain Name System (DNS). Its most prominent component, named (pronounced ''name-dee'': , short for ''name daemon''), performs both of the main DNS server roles, acting as an authoritative ...
to the solid phase of
silica Silicon dioxide, also known as silica, is an oxide of silicon with the chemical formula , most commonly found in nature as quartz and in various living organisms. In many parts of the world, silica is the major constituent of sand. Silica is o ...
under certain conditions.


Procedure

The stages of the method are lyse, bind, wash, and elute. More specifically, this entails the
lysis Lysis ( ) is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic (that is, "lytic" ) mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a ''lysate''. In molecular bio ...
of target cells to release nucleic acids, selective binding of nucleic acid to a silica membrane, washing away particulates and inhibitors that are not bound to the silica membrane, and elution of the nucleic acid, with the end result being purified nucleic acid in an aqueous solution. For lysis, the cells (blood, tissue, etc.) of the sample must undergo a treatment to break the
cell membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment (the ...
and free the nucleic acid. Depending on the target material, this can include the use of detergent or other buffers, proteinases or other enzymes, heating to various times/temperatures, or mechanical disruption such as cutting with a knife or homogenizer, using a mortar and pestle, or bead-beating with a bead mill. For binding, a
buffer solution A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is ...
is then added to the lysed sample along with
ethanol Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an alcohol with the chemical formula . Its formula can be also written as or (an ethyl group linked to a h ...
or
isopropanol Isopropyl alcohol (IUPAC name propan-2-ol and also called isopropanol or 2-propanol) is a colorless, flammable organic compound with a pungent alcoholic odor. As an isopropyl group linked to a hydroxyl group (chemical formula ) it is the simp ...
. The sample in binding solution is then transferred to a spin column, and the column is put either in a
centrifuge A centrifuge is a device that uses centrifugal force to separate various components of a fluid. This is achieved by spinning the fluid at high speed within a container, thereby separating fluids of different densities (e.g. cream from milk) or ...
or attached to a vacuum. The centrifuge/vacuum forces the solution through a silica membrane that is inside the spin column, where under the right ionic conditions, nucleic acids will
bind BIND () is a suite of software for interacting with the Domain Name System (DNS). Its most prominent component, named (pronounced ''name-dee'': , short for ''name daemon''), performs both of the main DNS server roles, acting as an authoritative ...
to the silica membrane, as the rest of the solution passes through. With the target material bound, the flow-through can be removed. To wash, a new buffer is added onto the column, then centrifuged/vacuumed through the membrane. This buffer is intended to maintain binding conditions, while removing the binding salts and other remaining contaminants. Generally it takes several washes, often with increasing percentages of ethanol/isopropanol, until the nucleic acid on the silica membrane is free of contaminants. The last 'wash' is often a dry step to allow the alcohol to evaporate, leaving only purified nucleic acids bound to the column. Finally, elution is the process of adding an aqueous solution to the column, allowing the
hydrophilic A hydrophile is a molecule or other molecular entity that is attracted to water molecules and tends to be dissolved by water.Liddell, H.G. & Scott, R. (1940). ''A Greek-English Lexicon'' Oxford: Clarendon Press. In contrast, hydrophobes are ...
nucleic acid to leave the column and return to solution. This step may be improved with salt, pH, time, or heat. Finally, to capture the eluate/eluent, the column is transferred into a clean microtube prior to a last centrifugation step.


Related methods

Even prior to the nucleic acid methods 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 Algae ( , ; : alga ) are any of a large and diverse group of photosynthetic, eukaryotic organisms. The name is an informal term for a polyphyletic grouping that includes species from multiple distinct clades. Included organisms range from ...
called
diatoms A diatom (Neo-Latin ''diatoma''), "a cutting through, a severance", from el, διάτομος, diátomos, "cut in half, divided equally" from el, διατέμνω, diatémno, "to cut in twain". is any member of a large group comprising sev ...
which shield their cell walls with
silica Silicon dioxide, also known as silica, is an oxide of silicon with the chemical formula , most commonly found in nature as quartz and in various living organisms. In many parts of the world, silica is the major constituent of sand. Silica is o ...
. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions. This was later improved using 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. For ease of handling, the use of glass beads was later changed to silica columns. And to enable use of automated extraction instruments, there was development of silica-coated paramagnetic beads, more commonly referred to as "magnetic bead" extraction.


See also

*
DNA separation by silica adsorption DNA separation by silica adsorption is a method of DNA separation that is based on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH conditions. Operations In order to conduct DNA separation by silica ...
* Guanidinium thiocyanate-phenol-chloroform extraction * Ethanol precipitation * SCODA DNA purification * Plasmid preparation


References

{{Molecular Biology Biochemistry methods Molecular biology