GENERAL PRINCIPLES OF DNA EXTRACTION

10 Apr

GENERAL PRINCIPLES OF DNA EXTRACTION

are sodium dodecyl sulfate (SDS), Tween 20, Triton X-100 and Nonidet P-40. The lysis buffer destabilizes the cell membranes, leading to the breakdown of cellular structure. The addition of a chaotropic salt, for example 6-M guanidine thiocyanate [9] or 6-M sodium chloride, during or after cell lysis, disrupts the protein structure by interfering with hydrogen bonding, Van der Waals interactions, and the hydrophobic interactions. Cellular proteins are largely insoluble in the presence of the chaotropic agent and can be removed by centrifugation or filtration. The reduced solubility of the cellular protein is caused by the excess of ions in the high concentration of salt competing with the proteins for the aqueous solvent, effectively dehydrating the protein. Commonly used commercial kits, for example, the Qiagen kits, exploit the salting-out procedure; the methods to isolate the DNA after the cellular disruption vary widely. Several DNA extraction methods are based on the binding properties of silica or glass particles. DNA will bind to silica or glass particles with a high affinity in the presence of a chaotropic salt [9, 10]. After the other cellular components have been removed the DNA can be released from the silica/glass particles by suspending them in water. Without the chaotropic salt the DNA no longer binds to the silica/glass and is released into solution. The silica method in particular has been shown to be robust when extracting DNA from forensic samples [1]; it is also amenable to automation [2, 3]. The advantage of the silica based salting-out methods are that they yield high molec- ular weight DNA that is cleaner than DNA from Chelex® 100 extractions. As with Chelex® 100 extractions, no highly toxic chemicals are involved. The process takes longerthantheChelex® 100andinvolvesmorethanonechangeoftubeandsoincreases the possibility of sample mixing and cross-contamination.

Phenol-chloroform-based DNA extraction

The phenol-chloroform method has been widely used in molecular biology but has been slowly phased out since the mid 1990s, largely because of the toxic nature of phenol. It is still used in some forensic laboratories; in particular it is still widely used for the extraction of DNA from bone samples and soils. Cell lysis is performed as in the previous method. Phenol-chloroform is added to the cell lysate and mixed and the phenol denatures the protein. The extract is then centrifuged and the precipitated protein forms a pellicle at the interface between the organic phenol-chloroform phase and the aqueous phase; this process is repeated two to three times or until there is no visible pellicle [11]. The DNA is then purified from the aqueous phase by ethanol precipitation or filter centrifugation. The method produces clean DNA but has some drawbacks: in addition to the toxic nature of phenol, multiple tube changes are required and the process is labour intensive.

FTA® paper
In Chapter 3 FTA®  paper was described as a method for sample collection and storage, particularly from buccal swabs and fresh blood samples. Once a sample is applied to

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