Review: Forensic Mitochondrial DNA Analysis: Current Practice and Future Potential, Part 1

Emily C. Lennert

Category: Biology

Keywords: DNA, mitochondrial, mtDNA, nuclear, nDNA, genetic, sequencing, Sanger, capillary electrophoresis, polymerase chain reaction, PCR

Article to be reviewed:

  1. Melton, T.; Holland, C.; Holland, M. “Forensic mitochondrial DNA analysis: Current practice and future potential.” Forensic Science Review. 2012, 24 (2), 101-122.

Additional references:

  1. Forensic DNA: Mitochondrial DNA. (Accessed September 27, 2016)
  2. mtDNA Basics. (Accessed September 27, 2016)
  3. Sanger Sequencing Methods. (Accessed September 27, 2016)

Disclaimer: The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.


This article discusses mitochondrial DNA as it pertains to forensic science. This review will focus on current practices in analysis, and a subsequent review of this article will address the future potential of this type of analysis.

Mitochondrial DNA (mtDNA) is DNA that is found in the mitochondria of a cell just outside of the nucleus. The mtDNA has a small, circular form, and most human cells contain hundreds of copies of mtDNA.1, 2, 3 This differs from nuclear DNA, which is the DNA found in the nucleus of a cell. Nuclear DNA (nDNA) contains genetic material from both parents; however, mtDNA contains only maternal genetic material, or genetic material originating from the mother. This allows a maternally related individual to provide a reference sample in instances where the individual in question is not available for DNA analysis, such as in identification of unknown human remains or remains that have been exposed to the environment for years.1, 3 Additionally, mtDNA is more likely to be recovered from small or degraded samples compared to nDNA, due to the presence of hundreds to thousands of copies of mtDNA per cell compared to only two copies of nDNA per cell.1, 3

Analysis of mtDNA is focused primarily on two regions, called hypervariable (HV) regions, which contain high levels of variation that can allow for discrimination among individuals or samples.3 In instances where HV1 and HV2 do not provide enough discriminating information, a third variable region, denoted as HV3, can be used. However, the authors of the review article note, mtDNA does not provide the same level of discriminating power as short tandem repeat (STR) analysis, which is performed on nDNA.1

Currently, according to the authors, mtDNA analysis is often performed on samples from old or cold cases, such as skeletal remains; on hairs that are less than 1 cm in length; or on non-human samples, such as canine hair. Hairs that are naturally shed, and therefore lacking the root, are also well suited for mtDNA analysis, even though they are not suited for nDNA analysis. However, no matter the specifics of the case, the methods for extraction, amplification, and sequencing of mtDNA are generally the same. Pre-extraction preparation is important to mtDNA analysis. Hairs must be thoroughly washed multiple times in an ultrasonic water bath, then rinsed with water and ethanol. Bone surfaces are sanded prior to cutting or powdering of an inner layer. Dilute bleach washes may be used on bone fragments and teeth to prepare them for extraction. After preparation, the DNA is extracted. An extraction solution is used, which contains an enzyme that breaks apart the cells, releasing the mtDNA into the extraction solution, which is a similar process to what is done for nDNA. Polymerase chain reaction (PCR) is used to amplify, or make copies of, the HV regions of mtDNA prior to analysis. After amplification, the DNA can be sequenced.

Sanger sequencing is a common method of sequencing for mtDNA and is considered the gold standard in DNA sequencing.1, 4 In Sanger sequencing, DNA fragments are separated by capillary electrophoresis. (See Fig. 1) Fluorescent dye is used to mark DNA fragments, with a different dye marking each base, and an optical detection device may be used to digitize the data.4 Mixtures present a similar challenge to mtDNA analysis as it does for nDNA analysis. Determination of mixture components is based on quantitative data to determine the major and minor contributors; however, Sanger sequencing does not provide truly quantitative data and is not useful for determination of major and minor contributors.


The authors state that mtDNA analysis appears well accepted in court. A subsequent review of the article, entitled “Review, Part 2: Forensic mitochondrial DNA analysis: Current practice and future potential” will discuss alternative methods and future potential for mtDNA analysis.

Scientific Highlights:

  • mtDNA is inherited from the mother. This allows for a maternally related individual to contribute a reference sample in instances where the individual is not available.
  • mtDNA is often recovered in instances where nuclear DNA cannot be recovered.
  • Sample preparation is a vital part of mtDNA analysis.
  • Sanger sequencing is the “gold standard” for mtDNA sequencing.Relevance: mtDNA analysis allows for analysis of samples which cannot be analyzed through nDNA sequencing due to degradation or lack of a sufficient amount of nDNA.Potential conclusions:

In samples or instances in which nDNA cannot be recovered, mtDNA allows for DNA sequencing to occur. mtDNA analysis is particularly important in old cases, cases in which evidence has degraded, or cases in which hair recovered is small or lacking a root.
Although not as discriminatory as nDNA, mtDNA still allows for discriminations among most samples due to HV regions.