dna and dna evidence 111enzyme Ta q polymerase is used to add nucleotides in a chain-like fashion
that faithfully follows the code established by the single-stranded template.^11
Thus, wherever there is a thymine (T) in the template strand Ta q will add
an adenine (A), and wherever a guanine (G) is encountered its cytosine (C)
counterpart will be added. This step-wise replication of the template strand
starts at one primer and continues nucleotide by nucleotide until the ending
primer is reached.^11 This completes the first cycle of PCR, and as each sub-
sequent cycle is repeated with denaturation, annealing, and extension, the
previous amount of DNA is doubled, which continues with each subsequent
cycle.9–11 The millions of copies of targeted DNA that constitute the product
of PCR are called amplicons.
7.3.3 Analysis (Electrophoresis, Detection, and Interpretation)
The principle of electrophoresis as used in the forensic analysis of human
DNA is applied whether the sample is undergoing the short tandem repeat
(STR) fragment analysis of autosomal or sex chromosomes or the direct
sequencing of the mitochondrial genome. In an aqueous environment, the
DNA molecule is negatively charged. Thus, in a polarized electrical field
and proper medium, the DNA will move toward the positive electrode. As
a general rule, the smaller fragments will move faster than the larger ones,
allowing the analyst to separate fragments of DNA according to size9,15
The electrophoresis matrix that is used by most laboratory systems is a
viscous polymer. This gel may be poured into an external slab or run inside
a single or multiple capillaries (an array). The older slab gel configuration
risked bleed-over from one injection lane to the next and took much longer
for a technician to prepare. The newer capillary arrays are cleaner and more
efficient but also more expensive (Figure 7.6).
Once the nucDNA is sorted or the mtDNA is sequenced by electro-
phoresis using a polyacrylamide gel matrix, the results must be visually
depicted in a way that can be analyzed and compared with other samples.
Detection of the separated amplicons varies between the STR analysis of
nuclear alleles and sequencing analysis conducted on areas of interest in the
mitochondrial genome.
In STR analysis, the pieces of DNA are tagged with fluorescent labels
during the amplification process.^11 Later, the sample is injected into the cap-
illary instrument or added to the slab gel and the segments pass through
a specific window. At that point a laser detects the fluorescent signal and
correlates it to a standard (allelic ladder) of known fragment sizes. The result-
ing DNA profile is a series of peaks that correlate to these fragments and are
sorted by size and dye color. Pairs of peaks usually indicate heterozygosity at
that location (locus) on the molecule, whereas a single peak generally indi-
cates the individual is homozygous or has only one variant at that locus.^16