Mitochondria, often thought of as powerhouses of the cell, are fascinating eukaryotic organelles with a double-layered membrane and their own genome. Mitochondrial DNA (Mt DNA) is typically about 16570 bases, circular, highly compact, haploid and contains 37 genes, all of which are essential for normal mitochondrial function. Thirteen of these genes provide instructions for making enzymes involved in oxidative phosphorylation, a process that uses oxygen and simple sugars to create adenosine triphosphate (ATP), the cell’s main energy currency. The remaining genes code for transfer RNA (tRNA) and ribosomal RNA (rRNA) which are necessary for translating messenger RNA transcribed from nuclear DNA, into protein molecules.
One of the most important characteristics of mitochondrial genome that is relevant to field of forensics is the copy number. Mitochondrial division is disassociated from cellular division and is stimulated by energy demand, so cells with an increased need for energy contain greater numbers of these organelles than cells with lower energy needs. Most human cells contain hundreds of copies of mt DNA genomes, as opposed to two copies of the DNA that is located in the nucleus. This high copy number increases the likelihood of recovering sufficient DNA from compromised DNA samples, and therefore, mt DNA analysis can play an important role in missing persons investigations, mass disasters, and other forensic investigations involving samples with limited biological material. Samples such as muscle, bone, hair, skin, blood and other body fluids, even if drastically degraded by environment or time, may still hold enough material for mt DNA typing.
Mt DNA provides valuable forensic DNA typing in certain circumstances. Mt DNA analysis can be used in situations where an individual is missing or unable to provide a biological sample and a living maternal relative is available to provide the necessary DNA sample for comparison purposes. Mt DNA is maternally inherited therefore all of the individuals in the maternal lineage of a family (i.e. mother, daughter, sibling) will share the same mitochondrial DNA. The mitochondria of each new embryo come from the mother’s egg, and all mothers have the same mitochondrial DNA as their children. The father’s sperm contributes only nuclear DNA. Comparing the Mt DNA profile of unidentified remains with the profile of a potential maternal relative can be an important technique in missing person investigations. However, the disadvantage of lineage marker such as Mt DNA is the lower power of discrimination as there is no genetic shuffling with recombination. Also, family members have indistinguishable haplotypes unless mutations have occurred- however, mutation rate of mtDNA is about 5-10 times higher than that of nuclear DNA and therefore, may provide some discriminatory power. While autosomal STR loci have an extremely powerful discriminatory advantage for human identification, only certain portions of the control region of mt DNA are highly variable among individuals. Forensic analysis typically involves examination of the sequence variation within two hypervariable (HV) regions, HV1 and HV2. While laboratories may work with slightly different ranges, HV1 spans at least from position ~16024 to ~16365 and HV2 from position ~73 to ~340. Typically only 610 bases examined. While the traditional method of Sanger sequencing is labor intensive and time-consuming, new techniques such as massively parallel sequencing allow for whole mt DNA genome sequencing approach to obtain high-resolution mt DNA data, which can discriminate between closely related maternal lineages. Short nucleotide polymorphism (SNPs) which are present outside of the control region traditionally queried by forensic scientists can provide additional resolution and discriminatory power.
Mt DNA analysis process evaluation involves extracting mt DNA from evidence and reference samples, PCR amplification of the HV1 and HV2 regions, sequencing of amplicons using both strands and compare sequences. Upon analysis, if there are two or more nucleotide differences between the questioned and known samples, the samples can be excluded as originating from the same person or maternal lineage. The data is also compared to mt DNA database to determine haplotype frequency. The mt DNA population database is a compilation of differences in mtDNA control regions from a random collection of unrelated individuals of various ethnic backgrounds. Haplotype frequency is a measure of how commonly the sequence is present in a specific population and therefore, is a measure of discriminatory power.
Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible. However, due to high copy number, mitochondrial DNA (mt DNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shafts.
- NEAFS Y-mtDNA Workshop (Butler and Coble) Y-chromosome and mitochondrial DNA analysis. http://www.cstl.nist.gov/strbase/pub_pres/NEAFS2006_YmtDNA_mtDNA1.pdf
- Templeton, et al. (2013) DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification. Investigative Genetics. 4, 26.
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