Biology Reference
In-Depth Information
Quantification of DNA
After extracting DNA an accurate measurement of the amount of DNA and also the
quality of the DNA extract is desirable. Adding the optimum amount of DNA to a
PCR will produce the best-quality results in the shortest time; adding too much or not
enough DNA will result in a profile that is difficult or even impossible to interpret.
This is especially important when profiling forensic samples, when it is very difficult
to know the state of preservation of the biological material and also, in many cases, it
is difficult to estimate how much cellular material has been collected. It is less impor-
tant to quantify DNA when using some reference samples - where similar amounts
of DNA can be expected to be extracted each time as there are not very many vari-
ables. Even so, many laboratories will still quantify the DNA from reference samples
as part of their standard analysis. In response to the importance of quantification of
samples recovered from the scene of crime, the DNA Advisory Board in the USA
adopted rules that made quantification of human DNA mandatory [46].
The quantity of DNA that can be extracted from a sample depends very much on
the type of material. Each nucleated cell contains approximately 6 pg of DNA: liquid
blood contains 5000
10 000 nucleated blood cells per millilitre; semen contains
on average 66 million spermatozoa per millilitre (the average ejaculation produces
2.75 ml of semen) [47]. Biological samples recovered from the scene of crime are
not usually in pristine condition and can often consist of a very small number of
shed epithelial cells; consequently, the amount of DNA that can be recovered can
be extremely low and difficult to quantify.
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Visualization on agarose gels
A relatively quick and easy method for assessing both the quantity and the quality of
extracted DNA is to visualize it on an agarose gel. Agarose is a polymer that can be
poured into a variety of gel forms - mini-gels approximately 10 cm long are sufficient
to visualize DNA. The gel is submerged in electrophoresis buffer and the DNA
solution is loaded into wells that are formed in the gel by a comb; an electric current
is applied across the gel and the negatively charged DNA migrates towards the anode.
The agarose gel forms a porous matrix and smaller DNA molecules move through the
gel more quickly than do larger DNA molecules. Dyes that intercalate with the DNA
double helix, such as ethidium bromide [12], can be added to the gel either before or
after electrophoresis, the amount of intercalated dye is proportional to the quantity
of DNA. An alternative dye, 4
,6-diamidino-2-phenylindole (DAPI), can be added
directly to the DNA before electrophoresis. This migrates through the gel bound to
the minor groove of double-stranded DNA [48]. DNA is visualized by placing the
gel on a transilluminator that emits UV light at 260 nm. Quantification standards
can be run alongside the unknown samples to allow the DNA concentrations to be
estimated. In addition to showing the presence of DNA, the size of the extracted
DNA molecules can also be estimated. High molecular weight DNA can be seen as
