DNA extraction

From Wikipedia, the free encyclopedia For the various methods, see Nucleic acid methods. DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher.[1] Currently it is a routine procedure in molecular biology or forensic analyses.

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1 Basic procedure of DNA extraction 2 Special Types of DNA Extractions 3 Detecting DNA 4 See also 5 References 6 External links 7 Further reading

Basic procedure of DNA extraction
There are three basic and two optional steps in a DNA extraction:

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Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methodsblending, grinding or sonicating the sample. Removing membrane lipids by adding a detergent or surfactants which also serves in cell lysis. Removing proteins by adding a protease (optional but often done). Removing RNA by adding an RNase (almost always done). DNA purification from detergents, proteins, salts and reagents used during cell lysis step. The most commonly used procedures are: o Ethanol precipitation usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium acetate. o Phenol–chloroform extraction in which phenol denatures proteins in the sample. After centrifugation of the sample, denaturated proteins stay in organic phase while aqueous phase containing nucleic acid is mixed with the chloroform that removes phenol residues from solution. (Note: for DNA isolation in used phenol buffered to pH 8, RNA must be isolated using acidic phenol.) o Minicolumn purification that 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.

to produce a blue-colored compound. the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde. especially plasmids may be easily isolated by cell lysis followed by precipitation of proteins. 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. a pure sample of DNA has a ratio of 1. or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation. This procedure involves chemical hydrolysis of DNA: when heated (e. indigo and other fabric dyes or haemoglobin in blood samples from microorganisms with thick cellular wall. usually in the TE buffer. such as humic acid from soil. 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 concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations. Special Types of DNA Extractions Specific techniques must be chosen for isolation of DNA from some samples. After isolation. Detecting DNA Main article: Quantification of nucleic acids A diphenylamine (DPA) indicator will confirm the presence of DNA. most notably inhibitors of PCR.8 at 260/280 . for example yeast On the other hand. the reaction requires a deoxyribose sugar and therefore is specific for DNA. DNA absorbs UV light at 260 and 280 nanometres. or in ultra-pure water. 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. see ancient DNA samples containing inhibitors of subsequent analysis procedures. which prevents enzymes like DNase from degrading the DNA. the DNA is dissolved in slightly alkaline buffer.g. extrachromosomal DNA is generally easy to isolate. Typical samples with complicated DNA isolation are:    archaeological samples containing partially degraded DNA. which traps chromosomal DNA in insoluble fraction and after centrifugation. A Hirt DNA Extraction is an isolation of all extrachromosomal DNA in a mammalian cell. Under these conditions. plasmid DNA can be purified from soluble fraction. and aromatic proteins absorb UV light at 280 nm.Refinements of the technique include adding a chelating agent to sequester divalent cations such as. ≥95 °C) in acid. diphenylamine. Mg2+ and Ca2+. which reacts with the compound.

These procedures allow differentiation of the repeated sequences within the genome. running it on an agarose gel. and analysis.and is relatively free from protein contamination. DNA can be quantified by cutting the DNA with a restriction enzyme. identification. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1. staining with ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration. It is these techniques which forensic scientists use for comparison. . Using the Southern blot technique.8. this quantified DNA can be isolated and examined further using PCR and RFLP analysis.