Biology Reference
In-Depth Information
example dyes from clothing and humic acids from bone samples, can interfere with
the analysis.
Fluorescence spectrophotometry
Ethidium bromide or DAPI can be used to visualize DNA in agarose gels; these
are both examples of chemicals that fluoresce at much higher levels when they
intercalate with DNA. In addition to staining agarose gels, fluorescent dyes can also
be used as an alternative to UV spectrophotometry for DNA quantitation. A range
of dyes has been developed that can be used with fluorescent microplate readers and
these are very sensitive. The PicoGreen
dye is specific for double-stranded DNA
and can detect as little as 25 pg/ml of DNA [46]. When PicoGreen
binds to DNA
the fluorescence of the dye increases over 1000-fold; ethidium bromide in compari-
son increases in fluorescence 50 - 100-fold when it intercalates with double-stranded
DNA [46]. PicoGreen
is very sensitive and is a powerful technique for quantifying
total DNA; it does however have the drawback that it is not human specific.
Hybridization
Hybridization-based quantification methods have been widely used in forensic genet-
ics since the early 1990s, in particular a commercially available kit Quantiblot
(Applied Biosystems). Extracted DNA is applied to a positively charged nylon mem-
brane using a slot or dot blot process; the membrane is the exposed to a probe, that
is specific to human DNA. A commonly used target was the D17S1 alpha-satellite
repeat that is on human chromosome 17 in 500 - 1000 copies. The probe can be
labelled in a number of ways including colorimetric and chemiluminescent.
A series of standards is applied to the membrane, and comparison of the signal
from the extracted DNA with the standards allows quantification. The advantage of
hybridization-based methods is that the quantification is human specific; agarose gel
electrophoresis and spectrophotometry detect total DNA and forensic samples that
have been exposed to the environment for any length of time are prone to colonization
by bacteria and fungi.
The hybridization systems do suffer from a lack of sensitivity. For samples produc-
ing a negative result, in many cases it is still possible to generate a profile after PCR.
The analysis of the results is also subjective, leading different operators to come to
different conclusions. In addition to the limited sensitivity, the process is labour inten-
sive, taking approximately 2 hours to produce the blot. Hybridization-based methods
have been largely replaced by real-time PCR systems.
Real-time PCR
When generating a DNA profile, the PCR products are normally analysed at the
end point after 28 - 34 cycles. It is, however, possible to monitor the generation
