Review: Forensic Mitochondrial DNA Analysis: Current Practice and Future Potential, Part 2

Category: Biology

Keywords: DNA, mitochondrial, mtDNA, nuclear, nDNA, genetic, sequencing, Sanger, capillary electrophoresis, polymerase chain reaction, PCR, screening, linear array, expanded sequence, DNA mixtures, heteroplasmy, mass spectrometry, MS, electrospray ionization, ESI-MS, pyrosequencing, deep sequencing, next generation sequencing

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:
2. Ahmadian, A.; Ehn, M.; Hober, S. “Pyrosequencing: History, biochemistry and future.” Clinica Chimica Acta. 2006, 363 (1-2), 83–94.

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.

Author’s Note: Prior to reading this review, read the previous review, “Forensic mitochondrial DNA analysis: Current practice and future potential.” Part 1 provides the background information on mtDNA required for this review.


This article discusses mitochondrial DNA (mtDNA) as it pertains to forensic science. This review will focus on the future potential of mtDNA analysis; a previous review of the article addressed the current practice of this type of analysis, i.e. conventional mtDNA analysis.

Several alternative methods in mtDNA analysis are either in development or currently available. While conventional mtDNA sequence analysis is reliable and comprehensive, alternative methods may complement or improve current practice. Several alternative methods for mtDNA analysis currently exist, including: screening techniques, expanded sequence analysis, techniques for mixtures and heteroplasmy, mass spectrometric approaches, and pyrosequencing. Below are five alternative methods that can be used to provide more information on mtDNA sequencing.

  1. Screening Methods: Conventional mtDNA analysis, while effective, is a time intensive and costly process. Screening of samples can reduce expenses and time spent on full sequencing of unnecessary samples. The Roche Applied Science LINEAR ARRAY Mitochondrial DNA HVI/HVII Region-Sequence Typing Kit is a screening kit that has been adopted by some labs. The linear array kit targets 10 short sequences, encompassing 18 polymorphic sites. Polymorphic sites are areas within the genome that contain two or more alleles, or forms of a gene, at one locus, or site. The linear array uses polymerase chain reaction (PCR) to amplify the mtDNA, similar to conventional mtDNA analysis. The linear array result is a barcode- like profile, which can be used to exclude samples so that only potential matches are sent for full sequencing. A disadvantage of this technique is the amount of sample required. While conventional sequencing may be performed on mtDNA extracts from less than 1cm of hair, linear arrays require at least 2cm of hair for sampling. In instances with limited amounts of sample, especially if a full profile will be desired for potential matches, full sequencing would be considered more appropriate.
  2. Expanded Sequence Analysis: In the early 1990s, unpublished observations reported that common mtDNA sequence profiles within HV1 and HV2 were being encountered in samples of European Caucasian descent. When a common HV1 and HV2 sequence is encountered, the practitioner may no longer be able to differentiate between two samples. The first approach to overcoming this issue is to sequence a third segment in the control region, called HV3. A German study of matching HV1/HV2 samples showed that approximately 20% of samples were resolved by the HV3 approach. If the HV3 approach does not resolve the profiles, the analyst can expand sequencing to the coding region. The coding region of mtDNA is the region containing DNA that codes for proteins. In the coding region, 13 protein-coding genes exist, which contain over 11,000 nucleotides, many of which have variable sequences. Individuals that have matching HV1/HV2 sequences rarely match within the coding region as well.
  3. Mixtures and Heteroplasmy: “Heteroplasmy” refers to variations within in individual’s mtDNA. Mutations in some of the individual’s mtDNA can lead to an individual possessing more than one mtDNA profile. These mutations generally occur at low levels, and are difficult to detect by conventional mtDNA analysis, i.e. Sanger sequencing. Likewise, mixtures are considered nearly impossible to resolve by conventional mtDNA analysis. Denaturing gradient gel electrophoresis (DGGE) was developed to identify heteroplasmy in the HV1 region. The technique provides better detection of variations in mtDNA than Sanger sequencing. However, the technique is considered by many analysts to be too involved and difficult for routine use. An alternative method, denaturing high performance liquid chromatography (dHPLC) was developed to replace DGGE. Using dHPLC, identical HV1/HV2 sequences of individuals can be verified, and identified samples with minor deviations from the sequence, i.e. heteroplasmy, in order to resolve identical sequences. The ability of a technique to be used to identify heteroplasmy is indicative of potential application to mixtures; heteroplasmy is like a mixture, but originates from one individual rather than two or more.
  4. Mass Spectrometry: Mass spectrometry has been researched as a potential tool for identification of variants, either for analysis of mixtures or heteroplasmy, as well as a potential method for individual profiling. Electrospray ionization-mass spectrometry (ESI-MS) has been developed as a high resolution, automated method for MS analysis of mtDNA. After PCR amplification, the sample is purified through an automated system, then injected into the ESI-MS. Base composition is derived from mass data, and a full sequence is assembled via computer algorithms. ESI-MS was used in mtDNA analysis of 225 unidentified remains recovered from a mass grave dating back to World War I. Results for ESI-MS analysis were reported to be “as good or better than the Sanger sequencing results.”1 The ESI-MS method has been found to produce equivalent sequencing when compared to Sanger sequencing, except in instances with a small sequence stretch, such as nucleotide positions 16251-16253. ESI-MS is stated to have higher potential for differentiation of HV1 and HV2. Additionally, ESI-MS for mtDNA sequencing is a highly automated process, easy for the analyst to conduct, and performs well. However, the instrument is expensive to acquire, if the lab does not possess it. In addition, base composition profiles by ESI-MS do not exist in a database for comparison purposes.
  5. Pyrosequencing and Deep Sequencing: Pyrosequencing is a “sequencing-by-synthesis” method; it is an enzyme driven sequencing method that utilizes light detection for real time monitoring of DNA synthesis.2 As a new DNA strand is synthesized, light is emitted that corresponds to the nucleotide that has been added.2 Pyrosequencing has been used to detect mixtures and heteroplasmy, but has been reported to have relatively low levels of mixture detection, similar to Sanger sequencing. Deep sequencing is part of a next generation sequencing (NGS) method. The term “deep sequencing” refers to the practice of sequencing a region multiple times. Deep sequencing by NGS has potential to be applied to investigations involving mixtures and heteroplasmy. It is expected that, in the future, deep sequencing methods will be sensitive enough to detect low-level variations and be precise enough to identify the variation itself. (Author’s note: the article discussed in this review does not provide an explanation of NGS. For an explanation of NGS, refer to “Review: Next generation sequencing and its applications in genetic forensics.”)

Relevance: Many alternative methods exist in mtDNA analysis and, one day, some alternative methods may become more prominent in the field of mtDNA analysis.

Potential conclusions:

  • Several alternative methods for mtDNA exist, and each method has benefits and weaknesses.
  • ESI-MS has considerable potential for application to mtDNA analysis, especially in instances where high throughput analysis is desired.
  • For mixtures and cases of heteroplasmy, next generation sequencing (NGS) appears to be accepted as the most promising method.