- 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 methods-blending, grinding or sonicating the sample.
- Removing membrane lipids by adding a detergent or surfactants.
- Removing proteins by adding a protease (optional but almost always done).
- Removing RNA by adding an RNase (often done).
- Precipitating the DNA with an alcohol — usually ice-cold ethanol 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.
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.
Special Types of DNA Extractions
A Hirt DNA Extraction is an isolation of all extrachromosomal DNA 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.
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 curve 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 sequencing
- Polymerase chain reaction
- DNA fingerprinting
- Forensic DNA analysis
- Ethanol precipitation
- DNA structure
Molecular biology OverviewElementLinked Life EngineeringConceptTechnique
Wikimedia Foundation. 2010.
Look at other dictionaries:
DNA 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… … Wikipedia
DNA nanoball sequencing — is a high throughput sequencing technology that is used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Fluorescent probes bind to… … Wikipedia
Extraction de l'ADN — Extraction d ADN L extraction de l ADN est une technique permettant d isoler l ADN de cellules ou de tissus. L ADN ainsi extrait peut ensuite être utilisé pour des recherches de biologie moléculaire, telles que le séquençage, la PCR ou le clonage … Wikipédia en Français
DNA microarray experiment — steps involved in a microarray experiment (some steps omitted) For DNA microarrays in general, see DNA microarray. This is an example of a DNA microarray experiment, detailing a particular case to better explain DNA microarray experiments, while… … Wikipedia
DNA — For a non technical introduction to the topic, see Introduction to genetics. For other uses, see DNA (disambiguation). The structure of the DNA double helix. The atoms in the structure are colour coded by element and the detailed structure of two … Wikipedia
DNA profiling — Not to be confused with Full genome sequencing. Forensic science … Wikipedia
DNA microarray — A DNA microarray (also commonly known as gene chip, DNA chip, or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes… … Wikipedia
Extraction d'ADN — L extraction de l ADN est une technique permettant d isoler l ADN de cellules ou de tissus. L ADN ainsi extrait peut ensuite être utilisé pour des recherches de biologie moléculaire, telles que le séquençage, la PCR ou le clonage. Il existe… … Wikipédia en Français
Use of DNA in forensic entomology — Forensic science Physiological sciences … Wikipedia
Differential extraction — refers to the process by which the DNA from two different types of cells can be extracted without mixing their contents. The most common application of this method is the extraction of DNA from vaginal epithelial cells and sperm cells from sexual … Wikipedia