DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis.
Outline of a DNA extraction
There are three basic steps in a DNA extraction, the details of which may vary depending on the type of sample and any substances that may interfere with the extraction and subsequent analysis.
- Break open cells by grinding or sonication, and remove membrane lipids by adding a detergent.
- Remove celluar and histone proteins bound to the DNA, by adding a protease, by precipitation with sodium or ammonium acetate, or by using a phenol/chloroform extraction step.
- Precipitate DNA in cold ethanol or isopropanol, DNA is insoluble in alcohol and cling together, this step also removes salts.
Detecting DNA
A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95oC) 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 comapring to a standard curve of known DNA concentrations.
Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280nm is used as a measure of DNA purity. DNA absords UV light at 260 nm, and protein absorbs UV light at 280 nm; a pure sample of DNA should be relatively free of protein and the 260/280 ratio should be high. A DNA preparation that is contaminated with protein will have a lesser 260/280 ratio.
DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, and comparing the intensity of the DNA with a DNA marker of known concentration.
Uses for DNA
Extracted genomic DNA contains nuclear and mitochondrial DNA, if DNA is extracted from plant materal it will also contain chloroplast DNA. Each of these types of DNA has forensic, diagnostic and phylogenetic uses, and make use of the polymerase chain reaction (PCR) to obtain specific information from the DNA.
Purified DNA may also be used for studying DNA structure and chemistry, examining DNA-protein interactions, carrying out DNA hyrbridizations, and for cloning and sequencing.
See also
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