DNA extraction
Encyclopedia
DNA isolation is a routine procedure to collect DNA
for subsequent molecular
or forensic
analysis. There are three basic and two optional steps in a DNA extraction:
Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+
and Ca2+
, which prevents enzymes like DNase from degrading the DNA.
Cellular and histone
proteins bound to the DNA can be removed either by adding a protease
or by having precipitated the proteins with sodium
or ammonium acetate
, or extracted them with a phenol-chloroform
mixture prior to the DNA-precipitation.
If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.
in a mammalian cell. The Hirt extraction process gets rid of the high molecular weight nuclear DNA
, leaving only low molecular weight mitochondrial DNA
and any viral episomes present in the cell.
of known DNA concentrations.
Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm
is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8. ..
DNA can be quantified by cutting the DNA with a restriction enzyme
, running it on an agarose gel
, staining with ethidium bromide
or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.
Using the Southern blot
technique, this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison, identification, and analysis.
DNA Extraction Virtual Lab
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...
for subsequent molecular
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...
or forensic
Forensics
Forensic science is the application of a broad spectrum of sciences to answer questions of interest to a legal system. This may be in relation to a crime or a civil action...
analysis. There are three basic and two optional steps in a DNA extraction:
- Breaking the cellsCell (biology)The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
open, commonly referred to as cell disruptionCell disruptionCell disruption is a method or process for releasing biological molecules from inside a cell.- Choice of disruption method:The production of biologically-interesting molecules using cloning and culturing methods allows the study and manufacture of relevant molecules.Except for excreted molecules,...
or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding or sonicatingSonicationthumb|right|A sonicator at the [[Weizmann Institute of Science]] during sonicationSonication is the act of applying sound energy to agitate particles in a sample, for various purposes. In the laboratory, it is usually applied using an ultrasonic bath or an ultrasonic probe, colloquially known as...
the sample. - Removing membrane lipids by adding a detergent or surfactants.
- Removing proteinProteinProteins 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...
s by adding a proteaseProteaseA protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....
(optional but almost always done). - Removing RNARNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
by adding an RNase (often done). - Precipitating the DNA with an alcohol — usually ice-cold 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...
or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.
Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...
and Ca2+
Calcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
, which prevents enzymes like DNase from degrading the DNA.
Cellular and histone
Histone
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
proteins bound to the DNA can be removed either by adding a protease
Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....
or by having precipitated the proteins with sodium
Sodium acetate
Sodium acetate, CH3COONa, also abbreviated NaOAc, also sodium ethanoate, is the sodium salt of acetic acid. This colourless salt has a wide range of uses.-Industrial:...
or ammonium acetate
Ammonium acetate
Ammonium acetate is a chemical compound with the formula CH3COONH4 . It is a white solid, which can be derived from the reaction of ammonia and acetic acid...
, or extracted them with a phenol-chloroform
Phenol-chloroform extraction
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...
mixture prior to the DNA-precipitation.
If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.
Special Types of DNA Extractions
A Hirt DNA Extraction is an isolation of all extrachromosomal DNAExtrachromosomal DNA
Extrachromosomal DNA is DNA located or maintained in a cell apart from the chromosomes....
in a mammalian cell. The Hirt extraction process gets rid of the high molecular weight nuclear DNA
Nuclear DNA
Nuclear DNA, nuclear deoxyribonucleic acid , is DNA contained within a nucleus of eukaryotic organisms. In mammals and vertebrates, nuclear DNA encodes more of the genome than the mitochondrial DNA and is composed of information inherited from two parents, one male, and one female, rather than...
, leaving only low molecular weight mitochondrial DNA
Mitochondrial DNA
Mitochondrial DNA is the DNA located in organelles called mitochondria, structures within eukaryotic cells that convert the chemical energy from food into a form that cells can use, adenosine triphosphate...
and any viral episomes present in the cell.
Detecting DNA
A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95 °C) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curveStandard curve
A standard curve is a quantitative research tool, a method of plotting assay data that is used to determine the concentration of a substance, particularly proteins and DNA. It can be used in many biological experiments...
of known DNA concentrations.
Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm
Quantification of nucleic acids
In molecular biology, quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. Reactions that use nucleic acids often require particular amounts and purity for optimum performance...
is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8. ..
DNA can be quantified by cutting the DNA with a restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
, running it on an agarose gel
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
, staining with ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.
Using the Southern blot
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
technique, this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison, identification, and analysis.
See also
- DNA sequencingDNA sequencingDNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
- Polymerase chain reactionPolymerase chain reactionThe polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
- DNA fingerprinting
- Forensic DNA analysis
- 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 structureDNA structureIn molecular biology, the term double helix refers to the structure formed by double-stranded molecules of nucleic acids such as DNA and RNA. The double helical structure of a nucleic acid complex arises as a consequence of its secondary structure, and is a fundamental component in determining its...
External links
DNA Extraction Virtual Lab