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 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 molecular 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 longer than the Chelex®® 100 and involves more than one change of tube and so increases the possibility of sample mixing and cross-contamination.

Was this article helpful?

0 0
Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook

Post a comment