112 Forensic dentistryDirect sequencing of mtDNA is a slightly more complicated procedure.
Following PCR the regions of interest on the molecule are subjected to cycle
sequencing or dye terminator analysis.^10 The nucleotides are incorporated
into a growing strand of replicated DNA from PCR. But unlike traditional
PCR, the extension process is randomly halted through the addition of
dideoxynucleotides. These nucleotides are unique in that they are missing
the 3′-hydroxyl group that would normally permit the extension process to
continue. Instead, the incorporation of this dideoxynucleotide terminates the
extension process. Although normal deoxynucleotides are also present , only
the dideoxy terminators are fluorescently tagged with one of four dye colors
for adenine, guanine, cytosine, and thymine. Later, when each piece of DNA
passes through the laser window, the instrument detects each fluorescing
color and determines whether A, C, G, or T is the terminator nucleotide for
that strand. Subsequently, when the strands are sorted according to length, the
instrument will compute the actual sequence of the original amplicon.11,17
Although many of the laboratory processes can be automated, the one
step that still depends on the skill and experience of the DNA analyst is the
interpretation of the data. All the peaks that are generated by the instru-
ment actually represent an STR allele in the case of nucDNA or a mitochon-
drial base in the case of mtDNA. Most laboratories require a minimum
of two experienced analysts to review all these data prior to conclusions
being reported. It is also the exclusive purview of the analyst to compare
data between two samples, draw a conclusion, and to calculate the statistical
weight of the opinion.
Figure 7.6 an analyst working with the computer-controlled genetic analyzer
that differentiates pCr products from thermal cyclers and obtains the final dna
profile. (Courtesy of the armed Forces dna identification laboratory.)