dna and dna evidence 109
DNA present in the sample. Prior to moving to the amplification step, it is
prudent to determine how much human DNA is actually present so this can
be targeted in subsequent procedures using the correct volume and dilution.
Excessive quantities of DNA may overwhelm the amplification reaction and
interfere with the interpretation of the final data. In the opposite extreme,
insufficient template DNA may provide only a partial profile or no profile
at all.^9 Several methods have been used to determine the concentration of
DNA extracted from the evidence, a step called quantitation. These methods
include the older slot blot assay and the more modern real-time quantitative
polymerase chain reaction (PCR) assay.
The slot blot assay uses a forty-nucleotide probe that binds to a specific
site of interest on human chromosome 17; the amount of bound probe is pro-
portional to the amount of DNA present. A subsequent chemiluminescent
(light-producing) reaction is used to expose x-ray film. Then a comparison
of the size and intensity of the “blots” on the film are compared with known
standards to assist the analyst to determine the quantity of human DNA that
is present in the sample. Although specific for primates, this test does not
reveal the quality or condition of the DNA that is present in the sample.
Real-time PCR quantitation measures the incremental increase in prod-
uct after each amplification cycle. During the extension phase of PCR, a
fluorescent reporter dye is released and activated. Based on the increasing
Figure 7.4 examples of biological fumehoods that are employed during dna
extraction procedures to minimize dna contamination and protect analysts
from organic extraction chemicals. (Courtesy of the armed Forces dna i dentifi-
cation laboratory.)