Conventional Forensic Markers And Methods Used In mtDna Testing

As mentioned earlier, the most extensive mtDNA variations between individuals in the human population are found within the control region, or displacement loop (D-loop). Two regions within the D-loop known as hypervariable region I (HV1, HVI, or HVS-I) and hypervariable region II (HV2, HVII, or HVS-II) are normally examined by PCR amplification followed by sequence analysis. Approximately 610 bp are commonly evaluated - 342 bp from HV1 and 268 bp from HV2 (Figure 10.3). The DNA sequence for each sample between nucleotide positions 16024 and 16365 in HV1 and 73 and 340 in HV2 is determined and then compared to the Anderson or the revised Cambridge Reference Sequence (as mentioned earlier, these reference sequences are equivalent for the control region). Differences are noted and reported with the nucleotide position and the altered base. Sometimes a third hypervariable region (HVIII) is examined that is 137 bp long and spans nucleotide positions 438-574. Additional polymorphic sites within HVIII can sometimes help resolve indistinguishable HVI/HVII samples (Lutz et al. 2000, Bini et al. 2003).

A number of different PCR and sequencing primers have been used to generate the DNA sequence data for HV1 and HV2. Various primer combinations will be discussed in the next section. The mtDNA control region has been estimated

1 VR1 1

342 bp

16569/1 1

268 bp

137 bp




16024 16365 ^ 73 ^ 340 438 574

16024 16365 ^ 73 ^ 340 438 574

F15989 PSI (263 bp)

F15 PSIII (271 bp)




PSII (221 bp)

F155 PSIV (227 bp)

AFDIL primer set

to vary only about 1-2% (7-14 nucleotides out of the 610 bases examined is different) between unrelated individuals (Budowle et al. 1999). This variation is scattered throughout the HV1 and HV2 regions and is therefore best measured with DNA sequence analysis. However, there are 'hotspots' or hypervariable sites and regions where most of the variation is clustered (Stoneking 2000). Several methods for rapidly screening mtDNA variation have been developed that may be used for excluding samples that do not match. These methods often focus on measuring variation at the hypervariable hotspots and include using sequence-specific oligonucleotide probes (Stoneking et al. 1991), mini-sequencing (Tully et al. 1996), and denaturing gradient gel electrophoresis (Steighner et al. 1999) as well as a restriction digest assay for HV1 amplicons (Butler et al. 1998a) and a reverse dot blot or linear array assay approach (Comas et al. 1999, Gabriel et al. 2003).

Figure 10.3 The three hypervariable (HV) regions of the mtDNA control region. HV1 spans nucleotide positions 16024-16365 (342bp), HV2 spans positions 73-340 (268 bp), and HV3, which is rarely examined in forensic testing, spans positions 438-574 (137bp). The general positions for variable regions VR1 and VR2 are noted although these are rarely used. PCR primer sets (PS) commonly used by the Armed Forces DNA Identification Laboratory (AFDIL) are illustrated. Primer nomenclature designates the 5'-nucleotide for each primer. PCR product sizes for each set of primers are noted in parentheses. The bottom section shows 'mini-primer' PCR product sizes that are used with highly degraded DNA samples to enable greater recovery of sequence information (see Gabriel et al. (2001a)).


In the following section, we describe the methodologies used for determining the sequence contained in mitochondrial DNA. Several nice overviews of forensic mtDNA analysis have been published and may be consulted for further information on this topic (Holland and Parsons 1999, Budowle et al. 2003, Isenberg 2004, Edson et al. 2004).

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