DNA extraction
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Overview
DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis.
Outline of a DNA extraction
There are three like 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.
- Chelate divalent cations such as Mg2+ and Ca2+ to stop dnase enzymes functioning and degrading the DNA
- Break open cells by grinding or sonication, and remove membrane lipids by adding a detergent.
- Remove cellular 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 clings together; this step also removes salt.
- Wash the resulting DNA pellet with alcohol
- Solubilize the DNA in a slightly alkaline buffer
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 comparing 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 absorbs UV light at 260 nm, and protein absorbs 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 and identification.
Uses for DNA
Extracted genomic DNA contains nuclear and mitochondrial DNA. If DNA is extracted from plant material it will also contain chloroplast DNA. Purified DNA may also be used for studying DNA structure and chemistry, examining DNA-protein interactions, carrying out DNA hybridizations, and for cloning and sequencing.