5.7.3 Automated and kit-based extraction of nucleic acids
Most of the current reagents used in molecular biology and the most common
techniques can now be found in kit form or can be automated, and the extraction of
nucleic acids by these means is no exception. The advantage of their use lies in the
fact that the reagents are standardised and quality control tested providing a high
degree of reliability. For example glass bead preparations for DNA purification have
been used increasingly and with reliable results. Small compact column-type prepar-
ations such as QIAGEN columns are also used extensively in research and in routine
DNA analysis. Essentially the same reagents for nucleic acid extraction may be used in
a format that allows reliable and automated extraction. This is of particular use where
a large number of DNA extractions are required. There are also many kit-based
extraction methods for RNA; these in particular have overcome some of the problems
of RNA extraction such as RNase contamination. A number of fully automated nucleic
acid extraction machines are now employed in areas where high throughput is
required, e.g. clinical diagnostic laboratories. Here the raw samples such as blood
specimens are placed in 96- or 384-well microtitre plates and these follow a set
computer-controlled processing pattern carried out robotically. Thus the samples are
rapidly manipulated and extracted in approximately 45 min without any manual
operations being undertaken.5.7.4 Electrophoresis of nucleic acids
Electrophoresis in agarose orpolyacrylamide gelsis the most usual way to separate
DNA molecules according to size. The technique can be used analytically or prepara-
tively, and can be qualitative or quantitative. Large fragments of DNA such as
chromosomes may also be separated by a modification of electrophoresis termed
pulsed field gel electrophoresis(PFGE). The easiest and most widely applicable
method is electrophoresis in horizontal agarose gels, followed by staining with
ethidium bromide. This dye binds to DNA by insertion between stacked base pairs
(intercalation), and it exhibits a strong orange/red fluorescence when illuminated
with ultraviolet light (Fig. 5.24). Very often electrophoresis is used to check the purity
and intactness of a DNA preparation or to assess the extent of an enzymatic reaction
during for example the steps involved in the cloning of DNA. For such checks
‘minigels’ are particularly convenient, since they need little preparation, use small
samples and give results quickly. Agarose gels can be used to separate molecules
larger than about 100 bp. For higher resolution or for the effective separation of
shorter DNA molecules polyacrylamide gels are the preferred method.
When electrophoresis is used preparatively, the piece of gel containing the desired
DNA fragment is physically removed with a scalpel. The DNA may be recovered from
the gel fragment in various ways. This may include crushing with a glass rod in a
small volume of buffer, using agarase to digest the agarose leaving the DNA, or by the
process ofelectroelution. In this method the piece of gel is sealed in a length of
dialysis tubing containing buffer, and is then placed between two electrodes in a tank
containing more buffer. Passage of an electrical current between the electrodes causes168 Molecular biology, bioinformatics and basic techniques