Introduction
Because of advancements in technology and techniques, mitochondrial DNA (mtDNA) analysis has become a common method in forensic procedure. Polymorphisms in human mtDNA were first discovered in 1980 (Brown 1980) while a complete sequence was first achieved in 1981 (Anderson et al., 1981). MtDNA sequencing is frequently used in cases where biological samples are degraded or low in quality, instead of nuclear DNA (nDNA), because each cell contains more than 1000 copies of mtDNA per cell instead of only two per cell in the case of nDNA. This article will examine the background information and techniques of mtDNA analysis as well as several of its applications.

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Background Information
Mitochondrial DNA is located outside the nucleus of a cell in organelles known as mitochondria. The chief advantage of mtDNA is the fact that it is present at a rate of between 100 and several thousand times per cell, making it much easier to be typed than nuclear DNA (James and Nordby, 2005). This characteristic makes mtDNA a very useful tool in a sample that is either degraded or limited in quantity. Its typical sources include hair, bones, teeth and bodily fluid such as saliva, blood, and semen. Another feature of mtDNA is that it is maternally inherited so a sample can be collected from any member of the maternal lineage (Giles et al. 1980). The mtDNA for humans is ~16,569 base pairs and consists of two regions, the control region and the coding region.
The control region is located in the displacement loop (D-loop), and contains hypervariable regions 1, 2, and 3 (HVI, HVII, and HVIII). HVI and HVII are sufficiently polymorphic to allow two samples to be differentiated. However, HVIII is currently being assessed to determine its capacity for use in discriminating between individuals and could one day be used to increase the discerning power of mtDNA. The D-loop of mtDNA does not contain any functional genes, and accumulates mutations at a rate of up to 10 times that of nDNA (James and Nordby, 2005). See image below for a sample mtDNA structure.
The HVI region is approximately 341 base pairs (bp) in length while the HVII region is 267bp. The other regions in the mtDNA genome have been successfully analyzed but have not been frequently used in forensic works because they haven’t been proven sufficiently hypervariable.
Techniques of mtDNA Analysis
The methods for mtDNA typing have gradually changed over the past thirty years. The process has gradually changed protocols from low-resolution restriction length polymorphism (RFLP) analysis to sequence analysis of the HVII and HVII regions and is moving towards a complete sequence of the mtDNA genome for each case. The sequencing and comparison of the mtDNA can be broken down into four steps: Extraction, polymerase chain reaction (PCR) amplification, sequencing, and comparison.
Extraction
A biological sample contains main substances besides DNA. The purpose of extraction is to separate the other material from the DNA before it is examined. The sample is prepared and mixed with certain organic chemicals that lyse the cell membranes, separate proteins from the DNA, and then denature the proteins and destroy their structures to decrease their solubility. Using a phenol chloroform, the denatured proteins are removed. The DNA is then purified using ethanol precipitation and isolation by centrifuge.
PCR Amplification
The polymerase chain reaction is process similar to the one used by cells to copy their own DNA. By using this process, a relatively small number of copies could be multiplied into over a billion in around thirty cycles. PCR amplification is a three step process. First, the two mtDNA strands are denatured by heat at 94°C. This means the strands separate from each other into two equal and opposite strands. Second, the sample is cooled to 60°C and the primers bind to the DNA template, which gives a start sequence to the DNA polymerase in step three, which extends the primers by adding the respective nucleic acids to the base strand and completes the sequence, thus turning one copy of the DNA into two. By repeating this process, the number of copies increases exponentially.
Sequencing
The primary sequencing method in mtDNA analysis is known as the Sanger method, which is a six step process that builds off the PCR amplification. First, the removal of remaining Deoxyribonucleotide triphosphates (dNTP) and primers from PCR through spin filtration or enzyme digestion is required. The PCR quantity is then found to determine if there is enough product to sequence the DNA. Then, four different colored fluorescent dyes are attached to the four differenet ddNTPs. After that, the unincorporated dyes from the sequencing reaction are removed using spin filtration. The purified sequencing reaction is then diluted in foramide and separated through gel electrophoresis. Finally, the sequence analysis of each reaction performed is compiled and interpreted.
Comparison
After sequencing the HVI and HVII regions of the DNA, the sequences are confirmed with the forward and reverse strands for that sample. Differences are then noted from the revised Anderson sequence, which is the newest standard for mtDNA. Finally, the known and the unknown samples are compared with each other and then compared with the database to determine halotype frequency.
Applications
As previously stated, current applications of mtDNA analysis are best suited to DNA which is very old and highly degraded. Some of the applications in this include individual identification, evolutionary biology, and maternal lineage testing.
Individual Identification
MtDNA can play a considerable role in the identification of individuals in which the biological sample would be highly degraded, for example, identifying remains of unknown soldiers. In June, 1998, a DNA sample from one of the US tombs of the unknown was collected and compared to reference samples from seven different families who had lost a family member during the war. The results eliminated six of the families, and provided a positive identification on the final family. The results proved consistent and the soldier was identified as Air Force First Lieutenant Michael J. Blassie. In this case, the small, closed population allowed relative ease of identification. To date, the remains of around 150 soldiers from the Vietnam War and 1,200 from the Korean War have been reunited with their families.
Evolutionary Biology
There are two main hypotheses about human evolution and phylogenic trees trying to guess where the human race branches off from the rest of the animal kingdom and spread all over the world. The multiregional evolution suggests that modern humans evolved from Neanderthals and Homo erectus at the same time in different parts of the world. This hypothesis is supported by fossil evidence, particularly a gradual change in facial structure from earlier to modern humans.
The other hypothesis suggests a more recent African origin around 100,000 – 200,000 years ago, in which a small group of modern humans populated the rest of the world. This would have been done without mixing genetic material with other forms of humans. Because the human mitochondrial genome was one of the first to be completely sequenced, it took researchers a while to see the advantage of sequencing the entire genome. First, mutations in the D-loop occur at a rate 5-10 times faster than that of normal nDNA, and having the complete genome showed the same polymorphism on two different loci. Also, while the D-loop was changing at a high rate, mutations outside the D-loop were near zero, allowing the rate of evolution for the rest of the genome to be seen clearly and evenly between different complexes.
The data collected gives evidence to support the recent African origin theory. By finding the substitution rate between the sequences, it is possible to find dates at which the genetic material coincided with itself and thus makes it possible to find approximate dates and points on the phylogenic tree. From this information, the sequence of events in human evolution can be established.
The data suggests a severe population constriction around 180,000 years ago. (Brown, 1980) This evidence supports the recent African origin hypothesis while with the multiregional hypothesis you would expect to see this constriction at an older date.
Maternal Lineage
Because mtDNA testing can be done with a smaller quantity of DNA, it is well suited for maternity testing and forming a family tree. Right now, mtDNA is the most common form of DNA analysis performed to determine parental maternity testing today. Obviously, this is used in the legal system for the purpose of deciding custody battles.
MtDNA also has precedence in accurately performing historical identifications through maternal lineage. A famous case of this would most likely be the case in which Jesse James’ body was identified by using a comparison between his DNA and the great-great-granddaughter of his sister. This lineage would provide a positive match if in fact it was Jesse James’ remains, which in fact it was.
However, the James case was not without its faults. The initial attempts at mtDNA analysis of the bone specimens from the specimen were not successful. More, specifically, there was no product formation during the Polymerase Chain Reaction. The most likely cause of this type of error would be the poor condition the material was in.(Stone et al. 2001) After running several tests on the bones, teeth and hair of the remains, mtDNA was collected and analyzed from two molar teeth and two hair fibers. This just shows that while mtDNA analysis has the possibility for up to 100% exclusion, because of mix-ups, laboratory errors, contamination, and degradation, it’s not 100% accurate.
Conclusion
Because of its impact on the forensic community, mtDNA analysis has become a power discrimination tool in forensic science. The methods that have been developed over the past thirty years have been firmly founded in scientific research. Each tool used in the aid of sequencing the mtDNA has a specific purpose that plays an important part the analysis as a whole. As technologies evolve, the discriminating power goes up, as in the case of adding the HVIII loci to the standard, and the error rates go down because of new information regarding contamination prevention procedures and determining the presence of degraded sample. Finally, by applying all of these concepts to the forensic world, mtDNA analysis becomes a powerful tool that has the potential to trace lineages, prove maternity, and identify unknown persons.
References

Anderson, S., Bankier, A.T., Barrel, B.G., Bruijn, M.H.L., Courlson, A.R., Drouin, J., Eperon,I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A., Staden, R., and Young, I.G., 1981, Sequence and Organization of the Human Mitochondrial Genome.MRC Laborator of Molecular Biology, Hills Road, Cambridge, UK
Brown, W. M. 1980 Polymorphism in mitochondrial DNA of humans as revealed by restriction endonuclease analysis Proc. Nati. Acad. Sci. USAVol. 77, No. 6, pp. 3605-3609
Butler, J. 2005, Forensic DNA Typing. Elsevier Science pp.242-298
Giles, R.E., Blanc, H., Cann, H.M., and Wallace, D.C. .1980, Maternal inheritance of human mitochondrial DNA, Proc. Nati. Acad. Sci. USAVol. 77, No. 11, pp. 6715-6719,
Ingman, M. ,2001,Mitochondrial DNA Clarifies Human Evolution, http://www.actionbioscience.org/evolution/ingman.html Image Source: MtDNA. http://www.nfstc.org/pdi/Subject09/images/pdi_s09_m02_01_a.1_large.jpg
James, S.H. and Nordby, J.J. 2005, Forensic Science: an introduction to scientific and investigative techniques. N.W. Ciroirate Blvd., Boca Raton, Florida
Michael Blassie Unknown No More, 2006, http://www.nlm.nih.gov/visibleproofs//galleries/cases/blassie.html
Stone A.C., Starrs J.E., Stoneking, M. Mitochondrial DNA analysis of the presumptive remains of Jesse James. J of Forensic Sci 2001;46(1):173-176.