Acid guanidinium thiocyanate-phenol-chloroform extraction: Difference between revisions
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'''Phenol-chloroform extraction''' is a [[liquid-liquid extraction]] technique in [[biochemistry]]. It is widely used in [[molecular biology]] for isolating [[DNA]], [[RNA]] and [[protein]]. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a [[Emulsion|biphasic mixture]]. This method may take longer than a [[Column-based nucleic acid purification|column-based system]] such as the silica-based purification, but has higher purity {{Fact|date=September 2008}} and the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short (<200 [[nucleotides]]) RNA species, such as [[siRNA]], [[miRNA]] and [[tRNA]]. |
'''Phenol-chloroform extraction''' is a [[liquid-liquid extraction]] technique in [[biochemistry]]. It is widely used in [[molecular biology]] for isolating [[DNA]], [[RNA]] and [[protein]]. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a [[Emulsion|biphasic mixture]]. This method may take longer than a [[Column-based nucleic acid purification|column-based system]] such as the silica-based purification, but has higher purity {{Fact|date=September 2008}} and the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short (<200 [[nucleotides]]) RNA species, such as [[siRNA]], [[miRNA]] and [[tRNA]]. |
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It was originally devised by Piotr Chomczynski and Nicoletta Sacchi and published in 1987 (referred to as ''Guanidinium thiocyanate-phenol-chloroform extraction'').<ref>{{cite journal | author = Chomczynski, P. & Sacchi, N. | title = Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction | journal = [[Anal. Biochem.]] | volume = 162 | pages = 156–159 | year = 1987 | doi = 10.1016/0003-2697(87)90021-2}}</ref><ref>{{cite journal | author = Chomczynski, P. & Sacchi, N. | title = Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: Twenty-something years on | doi = 10.1038/nprot.2006.83 | journal = [[Nature Prot.]] | volume =1 | pages = 581–585 | year = 2006}}</ref> It is sold by [[Sigma-Aldrich]] by the name [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=93289|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC TRI Reagent], by [[Invitrogen]] under the name [http://products.invitrogen.com/ivgn/product/15596026 |
It was originally devised by Piotr Chomczynski and Nicoletta Sacchi and published in 1987 (referred to as ''Guanidinium thiocyanate-phenol-chloroform extraction'').<ref>{{cite journal | author = Chomczynski, P. & Sacchi, N. | title = Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction | journal = [[Anal. Biochem.]] | volume = 162 | pages = 156–159 | year = 1987 | doi = 10.1016/0003-2697(87)90021-2}}</ref><ref>{{cite journal | author = Chomczynski, P. & Sacchi, N. | title = Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: Twenty-something years on | doi = 10.1038/nprot.2006.83 | journal = [[Nature Prot.]] | volume =1 | pages = 581–585 | year = 2006}}</ref> It is sold by [[Sigma-Aldrich]] by the name [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=93289|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC TRI Reagent], by [[Invitrogen]] under the name [http://products.invitrogen.com/ivgn/product/15596026 TRIzol] and by [http://www.bioline.com Bioline] as [http://www.bioline.com/h_prod_detail.asp?itemid=26 Trisure]. |
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==How it works== |
==How it works== |
Revision as of 16:42, 28 April 2010
![](http://upload.wikimedia.org/wikipedia/commons/thumb/f/fe/PhOH-CHCl3_extraction.svg/250px-PhOH-CHCl3_extraction.svg.png)
Phenol-chloroform extraction is a liquid-liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating DNA, RNA and protein. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a biphasic mixture. This method may take longer than a column-based system such as the silica-based purification, but has higher purity [citation needed] and the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short (<200 nucleotides) RNA species, such as siRNA, miRNA and tRNA.
It was originally devised by Piotr Chomczynski and Nicoletta Sacchi and published in 1987 (referred to as Guanidinium thiocyanate-phenol-chloroform extraction).[1][2] It is sold by Sigma-Aldrich by the name TRI Reagent, by Invitrogen under the name TRIzol and by Bioline as Trisure.
How it works
This method relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water-saturated phenol, chloroform and a chaotropic denaturing solution (guanidinium thiocyanate) resulting in an upper aqueous phase and a lower organic phase (mainly chloroform). Nearly all of the RNA is present in the aqueous phase, while DNA and protein partition in the interphase and organic phase, respectively. In a last step, RNA is recovered from the aqueous phase by precipitation with 2-propanol or ethanol. DNA will be located in the aqueous phase in the absence of guanidinium thiocyanate and thus the technique can be used for DNA purification alone.
Guanidinium thiocyanate denatures proteins, including RNases, and separates rRNA from ribosomes, while phenol, isopropanol and water are solvents with poor solubility. In the presence of chloroform or BCP (bromochloropropane), these solvents separate entirely into two phases that are recognized by their color: a clear, upper aqueous phase (containing the nucleic acids) and a bright pink lower phase (containing the proteins dissolved in phenol and the lipids dissolved in chloroform). Other denaturing chemicals such as 2-mercaptoethanol and sarcosine may also be used. The major downside is that Phenol and chloroform are both hazardous and inconvenient materials, and the extraction is often more laborious, so in recent years many companies now offer alternative ways to isolate DNA.
Reagents
- Phenol: The phenol used for biochemistry comes as a water-saturated solution with Tris buffer, as a Tris-buffered 50% phenol, 50% chloroform solution, or as a Tris-buffered 50% phenol, 48% chloroform, 2% isoamyl alcohol solution (sometimes called "25:24:1"). Phenol is naturally somewhat water-soluble, and gives a fuzzy interface, which is sharpened by the presence of chloroform, and the isoamyl alcohol reduces foaming. Most solutions also have an antioxidant, as oxidized phenol damages the nucleic acids. For RNA purification, the pH is kept around pH 4, which retains RNA in the aqueous phase preferentially. For DNA purification, the pH is usually near 7, at which point all nucleic acids are found in the aqueous phase.
- Chloroform: Chloroform is stabilized with small quantities of amylene or ethanol, because exposure of pure chloroform to oxygen and ultraviolet light produces phosgene gas. Some chloroform solutions come as pre-made a 96% chloroform, 4% isoamyl alcohol mixtures that can be mixed with an equal volume of phenol to obtain the 25:24:1 solution.
- Isoamyl alcohol: Some protocols include isoamyl alcohol as a stabilizing agent, while others do not require it at all.
See also
- Column-based nucleic acid purification
- Nucleic acid methods
- Ethanol precipitation
- DNA separation by silica adsorption
References
- ^ Chomczynski, P. & Sacchi, N. (1987). "Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction". Anal. Biochem. 162: 156–159. doi:10.1016/0003-2697(87)90021-2.
{{cite journal}}
: CS1 maint: multiple names: authors list (link) - ^ Chomczynski, P. & Sacchi, N. (2006). "Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: Twenty-something years on". Nature Prot. 1: 581–585. doi:10.1038/nprot.2006.83.
{{cite journal}}
: CS1 maint: multiple names: authors list (link)