Mitochondrial DNA (mtDNA) Sequencing
What is mitochondrial DNA sequencing?
Mitochondrial DNA sequencing, or mtDNA sequencing, is a process by which scientists analyze and compare samples of the DNA located in
the mitochondria of the body's cells. This technique is valuable because mitochondria can serve as a useful source of genetic material
when nuclear DNA samples are unavailable or degraded.
To learn more about how mtDNA sequencing is used for genetic and DNA testing,
visit the mtDNA Test page now.
How does it work?
Mitochondrial DNA is a small, circular DNA molecule found in mitochondria, which are small organelles found within all cells of the body.
Unlike nuclear DNA, which only exists inside the nucleus of a cell, mtDNA is found in multiple copies outside the nucleus.
This high concentration of mtDNA in cells makes it especially useful for testing samples that have low nuclear DNA concentration or are old and/or
degraded. For this reason, mtDNA is often used in forensic testing and other situations that require genetic
Mitochondrial DNA is almost exclusively inherited from the mother; less than 0.01% of a person's mtDNA is influenced by the father. Essentially, a mother will
pass an exact copy of her mtDNA on to her child. This inheritance pattern allows scientists to trace maternal lineages back through several generations.
mtDNA is composed of two regions arranged in a ring shape. These regions are the control (or non-coding) region and the coding region. The control
region is of most interest to scientists as it contains two variable segments, Hypervariable Region I (HVI) and Hypervariable Region II (HVII).
In humans, these regions have a higher mutation rate, such that even though a mother passes on the exact copy of mtDNA to her child, enough variation
has occurred through several generations that scientists can differentiate among different maternal lineages.
About mtDNA Sequencing
An mtDNA test uses sequencing, the process of determining the nucleotide sequences of a person's DNA. In a relationship test, the mtDNA sequences
of samples from two or more individuals are compared.
mtDNA sequencing is performed in 5 basic steps:
mtDNA Purification and Quantification
1. Sample preparation
When a sample for mtDNA testing is received, it first undergoes a preparation process. Any potential contaminants on the sample are removed.
Then the sample is placed in a solution that lyses the mitochondria, causing the mtDNA to be released.
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2. mtDNA Extraction
After the mitochondria are lysed, the mtDNA remains mixed in the sample solution. Additionally, unwanted biological molecules, such as proteins,
may still be attached to the mtDNA. To be separated from the excess molecules, the mtDNA is exposed to an organic chemical.
Once the mtDNA is cleaned of these molecules, the solution is separated and filtered, leaving the mtDNA completely free of the mitochondria and
excess biological molecules.
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3. PCR Amplification
Using PCR technology, the hypervariable regions of the mtDNA are copied and amplified. At the end of this step,
there are more than one billion copies of the mtDNA sample's hypervariable regions.
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4. mtDNA Purification and Quantification
The amplified DNA must be purified before it can be sequenced. A chemical is added to the mtDNA sample to remove any excess PCR
reagents remaining in the solution.
The sample is then quantified to determine the concentration of the amplified mtDNA regions. If the concentration is low, the sample will
undergo PCR again to increase the amount of amplified DNA. The sample will continue to undergo PCR and purification until a high concentration of
DNA is achieved and the sample is ready for sequencing.
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Sequencing of the mtDNA's hypervariable regions is a lot like the PCR technique. The sample is denatured, annealed,
and elongated in the same way as PCR, but additional molecules are present in the reactions. These molecules, called dideoxyribonucleotides, act like
the free nucleotides in a standard PCR solution; however, instead of promoting DNA strand growth, they stop it.
Specifically, an mtDNA double strand is denatured into two single strands. These strands are exposed to the PCR reagents (primers, nucleotide
bases, DNA polymerase, and buffer) and dideoxyribonucleotides (commonly called dideoxy bases). The dideoxy bases behave in the same way as the other
nucleotides in the PCR solution, but they contain a fluorescent tag and arrest DNA polymerization when incorporated into a growing DNA strand.
The strands start binding with nucleotides during elongation. Randomly (less than 5% of the time), the strand will bind with a dideoxy base
instead of a normal nucleotide and the elongation of the strand will stop. This results in a fragment of double-stranded DNA.
Gel electrophoresis is performed to separate the different fragments based on their size. The smaller
pieces of DNA will travel to the bottom of the gel.
The sequence of the mtDNA is determined based on the location of the fluoresced strand in the gel. The complete sequence of the mtDNA is often
automated, as the sequence is typically large.
See the image below for identifying the sequence based on location of the tags in the gel.
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Why use mitochondrial DNA sequencing?
As mentioned earlier, mtDNA sequencing is useful for obtaining genetic material from samples that have low concentrations of nuclear DNA or are
old or degraded.
More specifically, because mtDNA is highly preserved between generations, scientists use mtDNA testing for following genetic matrilineages
to study the migration of humans over time. mtDNA testing is also used to provide additional information in genetic reconstruction
cases and siblingship studies.
Visit the mtDNA Test page now to learn more.