A major advantage of mtDNA in terms of sequencing is that it is haploid and therefore only a single type exists (barring detectable heteroplasmy) for analysis. However, mixed samples from more than one biological source are commonly encountered in forensic settings. Generally speaking attempts are not made to decipher samples containing a mixture of more than one individual due to the complexity of the sequencing signals that could arise. Peak height ratios for two different bases cannot be used for reliable quantification of the two components because incorporation rates are not always even. Thus, the ratio of an A:G mixed base might be 50:50 at a particular position but when the complementary strand is sequenced a 70:30 or 80:20 ratio for the T:C bases might be observed because the polymerase incorporates the fluorescently-labeled ddTTP and ddCTP with different efficiencies than the A and G dideoxynucleotides.
If three or more sites within the 610 bases evaluated across HV1 and HV2 are found to possess multiple nucleotides at a position (i.e., sequence heteroplasmy), then the sample can usually be considered a mixture - either by contamination or from the original source material. Presently mixture interpretation is not attempted in forensic laboratories performing routine casework.
Some researchers are making pursuing efforts to resolve mtDNA mixtures through cloning and sequencing the resulting HV1/HV2 regions from individual colonies (Bever et al. 2003, Walker et al. 2004). Theoretically, each individual colony produced during the process of cloning corresponds to the control region from a single individual or a single component of heteroplasmy. Interpretation of mixtures is being attempted with statistical analysis from multiple clones. A number of pitfalls exist with this approach including the possibility of over-estimating the number of contributors due to the occurrence of heteroplasmic mitochondria. The number of contributors will be underestimated if individuals are closely related and members of the same mtDNA haplogroup (Walker et al. 2004). Denaturing HPLC has also been proposed as a possible approach to separating mtDNA amplicon mixtures (LaBerge et al. 2003), as has a mismatch primer-induced restriction site analysis method (Szibor et al. 2003b).
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