Before DNA can be profiled it must be extracted from it’s source material using one of a variety of extraction methods. Extraction is very important as the method chosen can influence the quality of the DNA obtained.
The most common methods of DNA extraction are:
Chelex is one of the oldest methods of DNA extraction and utilizes a chelating resin. It’s advantages are that it is cheap ,quick, has a low contamination rate and does not use any dangerous chemicals. However, it’s disadvantages include being inefficient for use on blood samples, producing low purity DNA samples and being unsuitable for restriction fragment length polymorphism DNA profiling.
Of all the methods of DNA extraction, the phenol- chloroform (also known as the organic method has been in use the longest. This is because it is the most effective at extracting the large amounts of high molecular weight DNA that were required for the RFLPs that created the first DNA fingerprints in the 1980s. It’s other main advantage is the fact that it can be used on a wide range of samples. However , this method does also have some disadvantages including being very labour intensive, being easily contaminated and exposing the scientist carrying out the extraction to dangerous chemicals.
N.B. DNA becomes soluble in the aqueous section as DNA is a polar compound and polar molecules dissolve better in polar solvents, (like water). Proteins are made up of amino acids, which may be polar or non polar. When exposed to Phenol, the folding of the proteins in the sample changes so that the non polar amino acids are on the outside of the molecule, making the protein more soluble in the phenol.
The FTA paper extraction method was initially used as a method of DNA collection in forensic science but due to the ease of the process has become a popular method of extraction. It’s other advantages include being easily repeated, easily automated and the added bonus that there is no need to quantify DNA extracted by FTA before PCR. However, due to the smaller nature of the “discs” of DNA obtained by this method, static electricity often causes them to jump out of their set location, leading to contamination.
Silica DNA extraction methods have become the staple for many labs as it is quick reliable and produces very high quality DNA. However, some DNA source materials such as chewing gum may interfere with the silica membrane.
To ensure that a good quality profile is obtained, it is important that the yield and purity of the sample is tested. Although there are four possible methods of determing the yeild of DNA obtained(1), the absorbance is most commonly used. DNA absorbance readings are taken at 260nm(A260) as this is where DNA most strongly abosorbs light.
To assess the total yield of DNA obtained from the extraction, you must first calculate the DNA concentration. This can be done using the absorbance of the sample as shown in the calculation below:
Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml
To obtain the yield you would then simply follow the calculation below:
DNA yield (µg) = DNA concentration × total sample volume (ml)
The purity of the DNA obtained is calculated using the A260/A280 absorbance ratio. A ratio of around 1.8 represents a pure DNA sample. (2)
1. The other methods that may be used to obtain DNA yeild are agarose gel electrophoresis, fluorescent DNA-binding dyes and a luciferase-pyrophosphorylation-coupled quantitation system.
2. This section of the article is adapted from the standard procedures for obtaining DNA yeild and purity freely available on www.promega.com
DNA extracts can then be used for down stream applications including:
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