Biology Reference
In-Depth Information
Nonidet P-40. The lysis buffer destabilizes the cell membranes, leading to the break-
down of cellular structure.
The addition of a chaotropic salt, for example 6 M guanidine thiocyanate [10]
or 6 M sodium chloride, during or after cell lysis, disrupts the protein structure
by interfering with hydrogen bonding, Van der Waals interactions and 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 com-
peting 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 [10, 11]. After the other cellular components have been
removed the DNA can be released from the silica/glass particles by suspending them
in water (Figure 4.2). In the absence of 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 [2]; it is also
amenable to automation [2 - 4].
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.
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 - 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 [12]. The DNA is then purified from
the aqueous phase by ethanol precipitation or filter centrifugation (Figure 4.3). 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
