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. Currently it is a routine procedure in molecular biology or forensic analyses. For the chemical method, there are many different kits are used for extraction, and selecting the correct one will save time on kit optimization and extraction procedures. PCR sensitivity detection considered to show the variation between the commercial kits.
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Basic procedure
There are three basic and two optional steps in a DNA extraction:
- Cells which are to be studied need to be collected.
- Breaking the cell membranes open to expose the DNA along with the cytoplasm within (cell lysis).
- Lipids from the cell membrane and the nucleus are broken down with detergents and surfactants.
- Breaking proteins by adding a protease (optional).
- Breaking RNA by adding an RNase (optional).
- The solution is treated with concentrated salt solution to make debris such as broken proteins, lipids and RNA to clump together.
- Centrifugation of the solution, which separates the clumped cellular debris from the DNA.
- DNA purification from detergents, proteins, salts and reagents used during cell lysis step. The most commonly used procedures are:
- 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.
- Phenol-chloroform extraction in which phenol denatures proteins in the sample. After centrifugation of the sample, denaturated proteins stay in the organic phase while aqueous phase containing nucleic acid is mixed with the chloroform that removes phenol residues from solution.
- Minicolumn purification that relies on the fact that the nucleic acids may bind (adsorption) to the solid phase (silica or other) depending on the pH and the salt concentration of the buffer.
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.
After isolation, the DNA is dissolved in slightly alkaline buffer, usually in the TE buffer, or in ultra-pure water.
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Special types
Specific techniques must be chosen for isolation of DNA from some samples. Typical samples with complicated DNA isolation are:
- archaeological samples containing partially degraded DNA, see ancient DNA
- samples containing inhibitors of subsequent analysis procedures, most notably inhibitors of PCR, such as humic acid from soil, indigo and other fabric dyes or haemoglobin in blood
- samples from microorganisms with thick cellular wall, for example yeast
Extrachromosomal DNA is generally easy to isolate, especially plasmids may be easily isolated by cell lysis followed by precipitation of proteins, which traps chromosomal DNA in insoluble fraction and after centrifugation, plasmid DNA can be purified from soluble fraction.
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.
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 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 a ratio of 1.8 at 260/280 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.
Source of the article : Wikipedia
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