Mitochondria is a double-layered membrane-bound organelle found in eukaryotic organisms. The organelle is also known as a powerhouse since it generates most of the cell’s adenosine triphosphate supply as an energy source.
DNA evidence samples that might be too little or degraded for Nuclear DNA analysis or miniSTR analysis. Mitochondrial DNA is inherited from one parent, and it does not undergo any genetic recombination except mutation. It provides only less genetic information than that of nuclear DNA. Mitochondrial DNA frequently provides information that nuclear DNA.
Each cell contains only two nuclear DNA chromosomes per cell, while mitochondrial DNA is five hundred to thousand per cell. The circular nature and the encapsulated structure inside the double-layered wall protect the mitochondrial DNA from enzymatic degradation. These factors enabled the analysis of mitochondrial DNA in determining the origin of bones, teeth, and ancient remains.
Mitochondrial chromosomes are circles of double-stranded DNA that contains 16569 base pairs. There is the asymmetric distribution of guanine present in the mt DNA strands. Most of the guanine present in one strand is the H (heavy) strand, and the other strand is known as the light (L) strand. The 16569 bp of mitochondrial DNA contains 37 genes that code for exons. There are no introns, and few nucleotides present between the coding regions. The mitochondrial genome contains a non-coding control region and origin of replication in one of the mtDNA strands. The replication starts in the H strand, and two hypervariable regions (HV1 and HV2) present in the mtDNA.
The methodology of Mitochondrial DNA Analysis
The copy number of Mitochondrial DNA is very high. More PCR cycles are necessary for mtDNA analysis. A tiny amount of Mitochondrial DNA is required for analysis and is prone to contamination. Purification of DNA is very significant in the analysis. Mitochondrial DNA extracted might get associated with PCR inhibitors.
Mitochondrial DNA sequencing methods
In 1996, the FBI began to examine mitochondrial DNA for forensic analysis. Sanger sequencing or Chain termination sequencing is used to sequence the hypervariable regions (HV1 and HV2). Each hypervariable region can be amplified using 34-38 PCR cycles instead of 28-30 PCR cycles as in nuclear DNA amplification. Melanin is a PCR inhibitor that can get coextracted along with hair samples. An extra amount of Taq polymerase should be added to the reaction to overcome PCR inhibition.
After sequencing, questioned sample and suspect samples are compared to the reference sequence, and differences found at specific sites are found. Human mitochondrial DNA was sequenced and is known as Anderson or Cambridge reference sequence (CRS). Sequence results are reported in terms of variation from the control region of the L strand. Only the base sequences deviated from the CRS are only reported, and others are the same. Software packages such as Sequencher can compare the question sample and the reference sequence next to each other.
[From Butler (2005), copyright ©2005 Elsevier Academic Press, Fig. 10.5.]
Screening Assays for mtDNA
The sequenced genome regions show the most variability between individuals, primarily in the hypervariable regions (HV1 and HV2) and hotspots. Most of the screening methods use sequence-specific oligonucleotides probes (SSO) in reverse dot blot and linear array assays. SSO probes examine the polymorphic sites in hypervariable regions through hybridization of PCR products.
Roche Applied Sciences developed a linear Array Mitochondrial DNA HVI/HVII Region-Sequence Typing kit. In this kit, SSO probes differentiate 19 polymorphic sites in 10 different positions within the control region. The time to finish this procedure is 6-8 hours. In this assay, there is four biotin-labeled primer mix to amplify HV1 and HV2 regions simultaneously.
Interpretation of Mitochondrial sequencing results
There are specific FBI guidelines to interpret mitochondrial sequencing results. Questioned DNA evidence samples are compared to reference sequences. If there two or more base sequence differences between questioned and known samples, then there is no common origin between the samples, and the sample is excluded. If there is a single sequence difference between the two samples, then the result is inconclusive. If there is no difference between the questioned and reference sample, then the questioned sample is not excluded. If there is heteroplasmy (Heteroplasmy is the presence of more than one mitochondrial DNA within a cell or individual. Heteroplasmy can also occur in sequence and length ) between the questioned and reference sample, then the sample cannot be excluded. If there is heteroplasmy at one strand and no changes in the other strand, then the sample is not excluded.