Chapter 23: Mitochondrial DNA Profiling

23.1: Human Mitochondrial Genome

• Mitochondria: Are cellular organelles that serve as the energy-generating components of cells.

  

Genetic Contents of Mitochondrial Organelle Genomes

• Cambridge reference sequence (CRS): The sequence was largely derived from a placental sample from an individual of European descent and also partially from HeLa cells and bovine cells.

• It was later discovered, by resequencing the original mtDNA sample, that the CRS contains substitution errors at 10 nucleotide positions.

  • The revised Cambridge reference sequence (rCRS) was published in 1999 and presented corrections to these substitution errors.

• Control Region: also known as Displacement Loop; it contains the origin of replication for one of the mtDNA strands but does not code for any gene products.

  

  

Maternal Inheritance of mtDNA

• Maternal inheritance is typically observed for the mtDNA genome, which is inherited differently from nuclear genes.
• The inheritance of the mtDNA genome does not obey the rules of Mendelian inheritance and is thus called non-Mendelian inheritance.
• Mitotype: Considered a a haplotype treated as a single locus.

23.2: mtDNA Polymorphic Regions

Hypervariable Regions

• The most polymorphic region of mtDNA is located within the D-loop.

• The three hypervariable regions in the D-loop are designated: Hypervariable Region I, Hypervariable Region II and, Hypervariable Region III

• The most common polymorphic regions of the human mtDNA genome analyzed for forensic purposes are HV1 and HV2.

  

  

Heteroplasmy

• Heteroplasmy occurs when an individual carries more than one mtDNA haplotype.

• Sequence heteroplasmy: The presence of two different nucleotides at a single position shown as overlapping peaks in a sequence electropherogram.

• Light heteroplasmy: Are often observed at the uninterrupted C stretches in sequencing, in which sequencing products with various lengths of polymeric cytosine residues are present.

  

23.3: Forensic mtDNA Testing

General Considerations

• mtDNA analysis is often used on samples derived from skeletal or decomposed remains.
• The surface of the sample should be cleaned to remove any adhering debris or contaminants.
• Bones and teeth are pulverized to facilitate extraction of the mtDNA Assay.
• mtDNA-specific quantization methods using real-time PCR can also be used to directly obtain measurements of mtDNA extracted.

mtDNA Screen Assay

• ASO Assay: It allows initial screening of mtDNA sequence polymorphisms and has the potential to reduce the number of samples required for mtDNA sequencing.
• Linear Array™ mtDNA HV1/HV2 region sequence typing kit: It utilizes reverse ASO configuration with a panel of immobilized ASO probes that detect common polymorphic sites.

mtDNA Sequencing

• A combination of PCR amplification and DNA sequencing techniques is utilized to reduce the time and labor needed to obtain DNA sequences from genomic DNA templates.

• mtDNA sequencing usually consists of:

  • PCR amplification;
  • DNA sequencing reactions;
  • Separation using electrophoresis; and
  • Data collection of Sequence analysis.

• DNA Sequencing Reactions

  • Chain-Termination or Sanger Method: An oligonucleotide primer that can anneal to a single stranded DNA template is utilized.
  • Cycle Sequencing: It utilizes thermal cycling to generate a single-stranded template for chain-termination sequencing reactions.

• Electrophoresis, Sequence Analysis, and Mitotype Designations

  • Reporting Format: Sequence differences relative to the rCRS are listed in data format.
  • Insertions: Described by noting the position followed by a decimal point and a number.
  • Deletions: These site designation is followed by letter d.
  • Hetero-plasmic Sites: The IUPAC codes for base calling can be applied to hetero-plasmic sites.

  

  

  

Interpretation of mtDNA Profiling Results

• Interpretation guidelines are used when comparing sequencing results between evidence and reference samples.
• Exclusion: If the sequences of questioned and known samples are different, then the samples can be excluded as originating from the same source.
  • It should be taken into account that higher mutation rates are found with the mtDNA genome than are found with the nuclear genome.
• Cannot Exclude: If the sequences are the same, the reference sample and evidence cannot be excluded as arising from the same source.
  • When an mtDNA profile cannot be excluded, it is desirable to evaluate the weight of the evidence.
• Inconclusive Result: If the questioned and known samples differ by a single nucleotide, and no heteroplasmy is present, the results are considered to be inconclusive.