110 Forensic dentistryintensity of the fluorescence from this dye, the real-time PCR instrument
plots the rate of accumulation of amplified DNA.^9 The point at which this
amplification curve crosses a predetermined amplification threshold is used
to form a standard curve. The analyst can determine the quantity of human
DNA in a given sample by finding where it falls along the standard curve.^9
Once the analyst measures the quantity of DNA in the sample, he or
she can proceed to PCR amplification (Figure 7.5). Not only does this pro-
cess replicate the target strand of DNA from the evidence, which ultimately
provides millions of copies of the original template to facilitate detection by
laboratory instruments, but the polymerase chain reaction can also be used
to target a specific area of interest for analysis, usually at the exclusion of all
other regions in the human genome.
The first step in PCR amplification is to denature or unwind the two
strands of the DNA molecule by elevating the temperature, usually to 94 to
96°C. This enables the single strands to be replicated individually. During the
second or annealing step the temperature is decreased, usually in the range of
55 to 72°C. This allows short segments of added DNA called primers to bind
in a complementary fashion at the ends of the target site of DNA from the
sample. In the third step, the temperature is elevated only slightly to 72 to 75°C
and extension begins.^10 In the correct chemical environment, the heat-stable
Figure 7.5 a bank of thermal cyclers used to perform pCrs on forensic samples
in a high-throughput forensic dna laboratory. (Courtesy of the armed Forces
dna identification laboratory.)