DNA EXTRACTION AND QUANTIFICATION

10 Apr

DNA EXTRACTION AND QUANTIFICATION

Figure 4.1 The Chelex® 100 extraction is quick and easy to perform. (a) The cellular material is added to 1 ml of TE (1 mM EDTA, 10 mM Tris: pH 8.0) and incubated at room temperature for 10-15 minutes. (b) The tube is centrifuged at high speed to pellet the cellular material and the supernatant is removed. (c) the pellet of cellular material is resuspended in 5% Chelex®, the tube is incubated at 56 ◦C for 15-30 minutes and then placed in a boiling water bath for 8 minutes. The tube is centrifuged at high speed for 2-3 minutes to pellet precipitated protein. (d) The supernatant contains the DNA and can be used directly in a PCR copolymers containing paired iminodiacetate ions [7].

The resin has a very high affinity for polyvalent metal ions, such as magnesium (Mg2+); it chelates the polyvalent metal ions and effectively removes them from solution.

The extraction procedure is very simple, the Chelex® 100 resin, which is supplied as beads, is made into a 5% suspension using distilled water. The cellular material is incubated with the Chelex® 100 suspension at 56 ◦C for up to 30 minutes.

Proteinase K, which digests most cellular protein, is often added at this point. This incubation is followed by 8-10 minutes at 100 ◦C to ensure that all the cells have ruptured and that the protein is denatured. The tube is then simply centrifuged to pellet the Chelex® 100 resin and the denatured protein at the bottom of the tube, leaving the aqueous solution containing the DNA to be used in PCR (Figure 4.1).

The Chelex® 100 suspension is alkaline, between pH 9.0 and 11.0, and as a result DNA that is isolated using this procedure is single stranded.

The major advantages of this method are: it is quick, taking around 1 hour; it is simple and does not involve the movement of liquid between tubes, thereby reducing the possibility of accidentally mixing samples; the cost is very low; and it avoids the use of harmful chemicals. Importantly, it is amenable to a wide range of forensic samples [7] . The DNA extract produced using this method is relatively crude but sufficiently clean in most cases to generate a DNA profile.

Silica Based DNA Extraction

Within molecular biology generally, the ‘salting out’ procedure has been widely used [8]. The first stage of the extraction involves incubating the cellular material in a lysis buffer that contains a detergent along with proteinase K. The commonly used detergents

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