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 DNA extraction

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. 

 

Basic methodology:

 

  1. DNA source material is added to a 5% Chelex suspension after incubation with Proteinase K.
  2. The sample is then exposed to 100 degree temperatures
  3. The mixture is then centrifuged to pull the Chelex to the bottom so the supernatant can be added directly to the PCR mix. 

 

Phenol - Chloroform extraction method

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.

 

Basic methodology:

 

  1. The DNA source material must be made soluble, this is achieved by adding it to an extraction buffer which contains EDTA to protect the DNA from nuclease degradation.
  2. Sodium dodecylsulphaye and proteinase K are added to denature the proteins with protect the DNA.
  3. The denatured proteins are then removed from the mix.
  4. The DNA is then purified with the addition of the phenol – chloroform and centrifuged. The DNA becomes soluble in the aqueous section of the organic aqueous mixture, whilst the debris remains in the organic section, so the DNA is isolated.

 

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. 

 

FTA paper based extraction method

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.

 

Basic methodology:

 

  1. Sample source (usually blood) is dropped onto the paper and as it dries the cells are lysed and the DNA becomes trapped within the matrix of the paper.
  2. The paper is punched to create discs, which are washed in a test tube.
  3. The discs are then washed with a solvent and added to the PCR mix

 

Silica based DNA extraction methods

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.

 

Basic methodology:

 

  1. Source material is added to a mixture containing both silica and chaotrophic salts which interfere with water molecules.
  2. In the presence of chaotrophic salts, DNA will bind to the silica rather than the disrupted water molecules.
  3. The contaminants present in the source material can then be washed away with salt, which in turn is washed away with Ethanol.
  4. The silica is then dried and DNA is eluted using water or a low salt buffer.

 

 

How to determine the purity and yield of DNA obtained

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)

Notes

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

Next Steps

DNA extracts can then be used for down stream applications including:

qPCR

PCR

DNA sequencing

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