Genetic analysis of mitochondrial DNA is one of the critical parts of forensic analysis because of its ubiquitous presence in biological material, even in the absence of nuclear DNA, and the chance of the presence of mitochondria is higher comparatively.
Before discussing the importance of mitochondria and analysis methods, it’s important to understand the basic structure and functions.
Characteristics and Structure of Mitochondria
Mitochondria are double membrane-bound cell organelles located in the cytoplasm of almost all types of human cells except Red blood cells (RBCs), which do not contain mitochondria. However, the number of mitochondria in each cell depends on its function.
Mitochondria share common features with many prokaryotic cells, such as similarities in size and structure, self-replicating genetic material such as DNA, and division by binary fission, which is also described in endosymbiont theory.
Mitochondrial Structure
The outer membrane is the smooth outer wall of the mitochondria that regulates the molecule’s movement across the mitochondria and cytoplasm.
The inner membrane is highly infolded and called cristae; these folds help increase the surface to perform reactions useful in ATP synthesis.
Inner membrane space is the space between the inner and outer membrane, which maintains the protein gradient for ATP synthesis.
The matrix is the innermost compartment of mitochondria; it is filled with a fluid-like substance ( some refer to it as a gel-like substance) where important biochemical reactions occur. Matrix is also the site where the mitochondrial DNA, ribosomes and enzymes for cellular respiration are present.
Mitochondrial Function
Various cellular processes like glycolysis, citric acid, and oxidative phosphorylation occur in mitochondria, which in turn produces ATP (Adenosine triphosphate). The cell’s energy currency due to this mitochondria is often referred to as the PowerHouse of the cell.
Mitochondria also regulate the calcium concentrations within cells and help various signalling mechanisms.
Mitochondria plays a key role in specific cellular processes such as apoptosis (Programmed cell death) by allowing the cytochrome c into the cytosol and further binding to Apaf-1 apoptotic protease activating factor 1 and initiation of further cascade mechanism.
Mitochondrial Genetics
Mitochondria are the cell organelles that have their extrachromosomal genome (mtDNA) which is much different from the nuclear genome mitochondrial DNA is located in the matrix ( a fluid filled cavity ) of the mitochondria along with ribosomes and enzymes involved in cellular respiration. Each mitochondrion has around 2 to 10 copies of this DNA.
Mitochondrial DNA was first identified and isolated from rat liver cells in 1963 by Margit Nass and Sylvan Nass. Still, the sequencing of the mitochondria was first done in the year 198, 18 years after its first discovery in rat liver cells.
This (mtDNA)is very similar to that of the bacterial genome, where the genome is double-stranded circular and lacks Histone octamers and introns. These two strands are named one heavy stand and the other a light chain; these are named after the density differences between the two strands.
MtDNA molecules are 16,569 base pairs long, with 37 coding sequences that code for
2 rRNA, 14 tRNA, and 13 polypeptides are essentially the enzymes involved in the oxidative phosphorylation reactions.
The D loop is considered the third strand of mtDNA located in the Non-coding region ( NCR ) of the mitochondria. The region in the D loop also consists of the two transcription promoters, one for each of the two strands.
Non-coding region NCR). The origin of replication is generally present within this region; the D loop comprises around 680 base pairs.
How do mitochondria serve the Forensic analysis?
The mutation rates within mitochondria are too high due to the lower fidelity of the mitochondrial DNA polymerase and lack of the repair machinery; thus, the mutation rate within the mitochondria is comparatively higher than that of nuclear DNA.
The most known regions for such mutations are observed in the regions known as hypervariable region 1 (HV1, positions 16 024 to 16 365) and in the hypervariable region 2 (HV2, positions 73 to 340). A third hypervariable region (HV3, positions 438 to 574).
These small regions with higher variability within the population are used for Forensic case investigations.
Every single mitochondrion can have 2-10 copies of mitochondrial DNA, and on average, a somatic cell may have up to 1000 mitochondria, so that shows that the availability of mitochondria is higher compared to that of nuclear DNA. This is helpful for forensic investigators when the nuclear DNA is degraded, or the extracted DNA quantity is minimal.
It’s also quite important to understand that MtDNA is exclusively maternally inherited, so all maternal relatives and siblings share the same haplotype, apart from the mutations. This unique character supports missing cases where one of the siblings or the relatives of maternal lineage can provide a reference sample of the analysis or case purpose.
Mitochondrial DNA (mtDNA) is exclusively inherited from the mother.
During the fertilisation process, the sperm cells contribute minimal cytoplasm to the zygote. Consequently, the majority of mitochondria within the fertilised egg originate from the maternal egg cell. Evidence also suggests that paternal mitochondria are actively degraded within the egg cell following fertilisation. This mechanism further emphasises the predominant role of maternal mitochondria in the developing embryo.
Heteroplasmy
It is necessary to understand Heteroplasmy in mitochondrial DNA before we go deep into the understanding of methods of forensic analysis.
Mitochondrial DNA shows a high degree of Heteroplasmy. The term mitochondrial Heteroplasmy refers to the presence of multiple variants of the mitochondrial DNA within a cell or an individual. Mitochondrial heteroplasmy can be caused by spontaneous mutations within the genome, bottleneck effect, and parental leakage, which is generally very rare.
In general, there are two classes of heteroplasmy that occur in the population: length polymorphisms and point polymorphism. However, most forensic laboratories worldwide report only point polymorphism. As a point, polymorphism provides much information related to human identification in forensic-related case works.
Even the guidelines made by the International Society for Forensic Genetics (ISFG) for human identification using mtDNA do not point to length polymorphism as mandatory information.
Types of Heteroplasmy in Mitochondria
mtDNA Heteroplasmy can manifest in various ways within an individual. Intra-tissue Heteroplasmy is the presence of multiple mtDNA types that can coexist within a single tissue. At the same time, In Tissue-Specific Heteroplasmy, an individual may exhibit Heteroplasmy in one tissue but have a single mtDNA type (homoplasmy) in another. In inter-tissue Heteroplasmy, the individual may possess distinct mtDNA types in different tissues. This scenario is generally considered a less frequent or rare type
The presence of this heteroplasmy made the use of the mtDNA much more complicated for the examination of DNA due to the lack of knowledge on the mechanism of heteroplasmy and the rate of heteroplasmy.
Methods for Mitochondrial DNA analysis
The methods of analysis used for mitochondrial DNA are evolving rapidly with advances in biology. In the earliest, i.e. in the 1980s, low-resolution fragment length polymorphism analysis was used to analyse the mtDNA. This analysis was performed using the 5 to 6 restriction endonucleases and is further followed up by PCR amplification.
The use of controlled portions of this genome for mitochondrial analysis came to acceptance after the 1990s for forensic DNA typing. Around the same time, the maximum variations in individuals are found in the D loop, known as a hypervariable region, as said earlier.
The standard methodology for the mtDNA analysis is mentioned here.
- MtDNA analysis by sequencing
- MtDNA analysis using RFLPS
DNA Extraction
In General, a wide variety of samples are received by forensic science labs for forensic analysis, includes samples of various body fluids such as blood, semen, menstrual fluids, vaginal fluid, saliva, urine, and whole organs such as intestine, stomach and liver, etc. but in a case where body fluids are not available other body tissues such as bones, teeth and hair are also used to obtain DNA.
Using evidence such as bones, teeth and hair to obtain DNA requires a lot of time and requires a lot of care while handling such evidence. Any physical changes can cause significant effects in further analysis. Forensic investigators generally approach MtDNA analysis when the availability of nuclear DNA is low or degraded.
Extraction methods depend on the type of sample from which DNA is being extracted various types of DNA extraction techniques are being used for this purpose, such as Silica-Based Membrane Extraction and chelex extraction used in isolating DNA from small samples. Organic extraction: A traditional method involving the use of organic solvents. Solid-phase extraction: DNA is bound to a solid matrix and then eluted.
PCR Amplification
Target Regions: In this step, the target regions are amplified up to 45 rounds, particularly the hypervariable regions (HVR-I and HVR-II). These regions exhibit high levels of variation among individuals, as mentioned in the genetics of mitochondria.
Sequencing
There are two common methods for sequencing DNA. The most known and traditional method is Sanger Sequencing. This is also known as the chain termination method, which incorporates the Dideoxynucleotides (ddNTPs) into the growing chain as per the ISFG guidelines for human identification using mitochondrial DNA analysis. Most forensic labs all over the world have started using the Sanger sequencing method to sequence the HV1 and HV2 regions in mtDNA, while some labs have started to study the regions of HV3 in recent years.
The use of next-generation sequencing such as Illumina is helpful for sequencing the whole mtDNA rapidly, which saves time and is helpful for faster forensic report producing.
Interpretation of results
After completion of mtDNA sequence analysis, a comparison of questioned (Q) and known (K) samples is done using the results of edited and reviewed sequences. A comparison of two sequences will give the result of a perfect match or not a match. If the samples match at each evaluated site, then they are termed as concordant.
Results are generally grouped into three categories:
i. Exclusion: If there are two or more differences in nucleotides between questioned and known samples, then it can be interpreted that the samples are not from the same source.
ii. Inconclusive: The result is considered inconclusive if there is a difference of one nucleotide between the known and questioned samples.
iii. Failure to exclude or cannot exclude: If there is a match between bases at each position or common length variant in the HV2 C-stretch of questioned and known samples, then it can be interpreted that the samples are from the same source or maternal lineage.
In some cases, the reference samples may not be available. In such cases, the forensic investigators depend on the databases that are already available.
Some of the most common databases are:
- MITOMAP (most commonly used database)
- GenBank
- EDNAP Mitochondrial DNA Population Database
RFLP Analysis
In this method, the obtained mitochondrial DNA after extraction is amplified using PCR amplification, and then it is treated with restriction enzymes; this treatment cleaves the DNA at restriction sites in multiple sites within the mtDNA. Due to the presence of variations in the mtDNA within the population, the location of the restriction sites varies from individual to individual, which in turn produces fragments of the DNA into various sizes. Using gel electrophoresis, these DNA fragments are analysed based on size.
Applications of Mitochondrial DNA and Uses of Mitochondrial DNA Analysis
Forensic science
Using mtDNA in forensic science investigations. The analysis of mtDNA has become crucial in forensic investigations as it can recover evidence from burnt remains, ancient carcasses, or decayed wildlife. Maternal lineage may also be determined through mitochondrial DNA sequencing
in events of kidnapping or when searching for missing individuals due to mtDNA being passed exclusively through mothers, this can be helpful
Evolutionary Biology and Anthropology
MtDNA provides knowledge on how humans evolved, the changes that people went through and the history of settlement of different nations and groups of humanity
Also useful in Phylogeography analysis
Medical Genetics
Mutations and errors in Mitochondrial DNA cause Some neurodegenerative, muscle, and metabolic diseases Such as Mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS) syndrome, Myoclonic epilepsy with ragged red fibers (MERRF), Neuropathy, ataxia and retinitis pigmentosa (NARP) syndrome, Leber hereditary optic neuropathy (LHON)
Limitations of Mitochondria DNA analysis
There are some limitations to using mitochondrial DNA for forensic purposes. A major limitation is that mitochondrial DNA provides limited information compared to nuclear DNA. For example, using mitochondrial DNA cannot provide much information about individuals, such as eye colour, hair colour, or any physical features.
The other limitation is that mtDNA analysis is much more time-consuming and expensive than nuclear DNA.
References
- “Mitochondrial DNA Common Mutation Syndromes.” Children’s Hospital of Philadelphia, https://www.chop.edu/conditions-diseases/mitochondrial-dna-common-mutation-syndromes. Accessed 17 January 2025.
- “Mitochondrial DNA in forensic use.” https://pmc.ncbi.nlm.nih.gov/articles/PMC8457767/.
- “Mitochondrial DNA in human identification: a review.” DOI 10.7717/peerj.7314.
- “Principles of Forensic DNA for Officers of the Court | The Two Types of DNA: Nuclear and Mitochondrial.” National Institute of Justice, 9 June 2023, https://nij.ojp.gov/nij-hosted-online-training-courses/principles-forensic-dna-officers-court/02-biology-dna/biological-terminology/two-types-dna-nuclear-and-mitochondrial. Accessed 17 January 2025.
- “Mitochondrial DNA in human identification: a review.” DOI 10.7717/peerj.7314.
