Phenol-chloroform extraction
Encyclopedia
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
. It is widely used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
for isolating DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
and protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a biphasic mixture
Emulsion
An emulsion is a mixture of two or more liquids that are normally immiscible . Emulsions are part of a more general class of two-phase systems of matter called colloids. Although the terms colloid and emulsion are sometimes used interchangeably, emulsion is used when both the dispersed and the...
. This method may take longer than a column-based system
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 to the solid phase depending on the pH and the salt content of the buffer, which may be a Tris-EDTA buffer or Phosphate buffer...
such as the silica-based purification, but has higher purity 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
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
, miRNA
Mirna
Mirna may refer to:geographical entities* Mirna , a river in Istria, Croatia* Mirna , a river in Slovenia, tributary of the river Sava* Mirna , a settlement in the municipality of Mirna in Southeastern Sloveniapeople...
, gRNA and tRNA. Column methods also shear large DNA fragments, which may or may not be a problem depending on downstream applications.
It was originally devised by Piotr Chomczynski and Nicoletta Sacchi and published in 1987 (referred to as Guanidinium thiocyanate-phenol-chloroform extraction). The reagent used specifically for RNA extraction is sold by Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich Corporation , is a life science and high technology company with over 7,600 employees and operations in 40 countries. Its chemical and biochemical products and kits are used in scientific research, biotechnology, pharmaceutical development, the diagnosis of disease, and as key...
by the name TRI Reagent, by Invitrogen
Invitrogen
Invitrogen Corporation was a large, multinational biotechnology company headquartered in Carlsbad, California. In November 2008, a merger between Applied Biosystems and Invitrogen was finalized...
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 phenolPhenol
Phenol, also known as carbolic acid, phenic acid, is an organic compound with the chemical formula C6H5OH. It is a white crystalline solid. The molecule consists of a phenyl , bonded to a hydroxyl group. It is produced on a large scale as a precursor to many materials and useful compounds...
, chloroform
Chloroform
Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous...
and a chaotropic denaturing solution (guanidinium thiocyanate
Guanidinium thiocyanate
Guanidinium thiocyanate is a chemical compound used as a general protein denaturant, being a chaotropic agent, although it is most commonly used in the extraction of DNA and RNA.Note: this compound may also be recognized as guanidine thiocyanate...
) resulting in an upper aqueous phase and a lower organic phase (mainly chloroform). Nucleic acid (RNA, DNA) partitions in the aqueous phase, while protein partitions in organic phase. In a last step, RNA is recovered from the aqueous phase by precipitation with 2-propanol or ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
. 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
Guanidinium thiocyanate
Guanidinium thiocyanate is a chemical compound used as a general protein denaturant, being a chaotropic agent, although it is most commonly used in the extraction of DNA and RNA.Note: this compound may also be recognized as guanidine thiocyanate...
denatures proteins, including RNases, and separates rRNA from ribosomes, while phenol
Phenol
Phenol, also known as carbolic acid, phenic acid, is an organic compound with the chemical formula C6H5OH. It is a white crystalline solid. The molecule consists of a phenyl , bonded to a hydroxyl group. It is produced on a large scale as a precursor to many materials and useful compounds...
, isopropanol and water are solvents with poor solubility. In the presence of chloroform
Chloroform
Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous...
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
2-Mercaptoethanol
2-Mercaptoethanol is the chemical compound with the formula HOCH2CH2SH. It is a hybrid of ethylene glycol, HOCH2CH2OH, and 1,2-ethanedithiol, HSCH2CH2SH...
and sarcosine
Sarcosine
Sarcosine, also known as N-methylglycine, is an intermediate and byproduct in glycine synthesis and degradation. Sarcosine is metabolized to glycine by the enzyme sarcosine dehydrogenase, while glycine-N-methyl transferase generates sarcosine from glycine. Sarcosine is a natural amino acid found in...
may also be used.
The major downside is that phenol
Phenol
Phenol, also known as carbolic acid, phenic acid, is an organic compound with the chemical formula C6H5OH. It is a white crystalline solid. The molecule consists of a phenyl , bonded to a hydroxyl group. It is produced on a large scale as a precursor to many materials and useful compounds...
and chloroform
Chloroform
Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous...
are both hazardous and inconvenient materials, and the extraction is often laborious, so in recent years many companies now offer alternative ways to isolate DNA.
Reagents
- Phenol: The phenol used for biochemistryBiochemistryBiochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
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 alcoholIsoamyl alcoholIsoamyl alcohol +P is a clear, colourless alcohol with the formula 2CHCH2CH2OH. It is one of several isomers of amyl alcohol. It is a main ingredient in the production of banana oil, an ester found in nature and also produced as a flavouring in industry....
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 ethanolEthanolEthanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
, because exposure of pure chloroform to oxygen and ultraviolet light produces phosgenePhosgenePhosgene is the chemical compound with the formula COCl2. This colorless gas gained infamy as a chemical weapon during World War I. It is also a valued industrial reagent and building block in synthesis of pharmaceuticals and other organic compounds. In low concentrations, its odor resembles...
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: Isoamyl alcohol may reduce foaming and ensure deactivation of RNase.
See also
- Column-based nucleic acid purificationColumn-based nucleic acid purificationColumn-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 to the solid phase depending on the pH and the salt content of the buffer, which may be a Tris-EDTA buffer or Phosphate buffer...
- Nucleic acid methodsNucleic acid methodsNucleic acid methods are the techniques used to study nucleic acids .Purification*Phenol-chloroform extraction*minicolumn purification*RNA extractionQuantification*Abundance in weight: spectroscopic quantification...
- Ethanol precipitationEthanol precipitationEthanol precipitation is a method used to purify and/or concentrate RNA, DNA and polysaccharides such as pectin and xyloglucan from aqueous solutions.- Theory :DNA is polar due to its highly charged phosphate backbone...
- DNA separation by silica adsorptionDNA separation by silica adsorptionDNA Separation by Silica Adsorption is an important method of DNA separation that is used in novel technologies that use micro-channels. The principle behind this type of separation relies on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH...