Mitochondrial DNA (World of Microbiology and Immunology)
Mitochondria are cellular organelles that generate energy in the form of ATP through oxidative phosphorylation. Each cell contains hundreds of these important organelles. Mitochondria are inherited at conception from the mother through the cytoplasm of the egg. The mitochondria, present in all of the cells of the body, are copies of the ones present in at conception in the egg. When cells divide, the mitochondria that are present are randomly distributed to the daughter cells, and the mitochondria themselves then replicate as the cells grow.
Although many of the mitochondrial genes necessary for ATP production and other genes needed by the mitochondria are encoded in the DNA of the chromosomes in the nucleus of the cell, some of the genes expressed in mitochondria are encoded in a small circular chromosome which is contained within the mitochondrion itself. This includes 13 polypeptides, which are components of oxidative phosphorylation enzymes, 22 transfer RNA (t-RNA) genes, and two genes for ribosomal RNA (r-RNA). Several copies of the mitochondrial chromosome are found in each mitochondrion. These chromosomes are far smaller than the chromosomes found in the nucleus, contain far fewer genes than any of the autosomes, replicate without going through a mitotic cycle, and their morphological structure is more like a bacterial chromosome than it is like the chromosomes found in the nucleus of eukaryotes.
Genes which are transmitted through the mitochondrial DNA are inherited exclusively from the mother, since few if any mitochondria are passed along from the sperm. Genetic diseases involving these genes show a distinctive pattern of inheritance in which the trait is passed from an affected female to all of her children. Her daughters will likewise pass the trait on to all of her children, but her sons do not transmit the trait at all.
The types of disorders which are inherited through mutations of the mitochondrial DNA tend to involve disorders of nerve function, as neurons require large amounts of energy to function properly. The best known of the mitochondrial disorders is Leber hereditary optic neuropathy (LHON), which involves bilateral central vision loss, which quickly worsens as a result of the death of the optic nerves in early adulthood. Other mitochondrial diseases include Kearns-Sayre syndrome, myoclonus epilepsy with ragged red fibers (MERFF), and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS).
See also Mitochondria and cellular energy; Mitochondrial inheritance; Ribonucleic acid (RNA)
Mitochondrial DNA Analysis (World of Forensic Science)
In human cells, DNA is found in both the nucleus and the mitochondria. The mitochondrion is an organelle responsible for the molecular products that provide the energy to the cell. There is a single nucleus in human cells and it contains two copies of DNA, one originating from the father and one from the mother. In contrast, there may be hundreds or thousands of mitochondria in human cells and the DNA in a single mitochondrion may be copied numerous times. Nuclear DNA is much longer than mitochondrial DNA, also written mtDNA, however the fact that there are so many more copies of mtDNA makes it extremely useful in cases in which there is only a small sample or the sample has been degraded. In addition, some biological materials such as hair shafts, teeth, and bones do not contain any cell nuclei, but mitochondria may be present and mitochondrial DNA analyses can be performed.
When a sperm fertilizes an egg, the DNA-containing head of the sperm fuses with the egg, but the tail and midsection are left on the outside of the egg. The mitochondria of a sperm are found in the tail and midsection as these parts require energy in order to propel the sperm. Because the mitochondria of the sperm never reach the inside of the egg, all the mitochondria in the embryo come from the egg. As a result the mitochondrial DNA in a child is identical to that of the mother. Mitochondrial DNA is therefore useful for proving maternal relationships in forensic investigations.
The DNA molecule is made up of a sequence of four different smaller molecules called nucleotides: adenine (A), guanine (G), cytosine (C) and thymine (T). DNA is a double stranded molecule and its nucleotides always associate themselves with a complementary nucleotide; if adenine is on one of the strands, thymine is across from it on the other strand. Similarly, if cytosine is on one strand, guanine will be found across from it on the other strand. Because the nucleotides of DNA are found in pairs on the two strands, the nucleotide sequence is also called a sequence of base pairs (bp).
Mitochondrial DNA is approximately 16,569 base pairs long and the genome is usually found in a ring-like conformation. There are two major parts of the molecule. A coding region accounts for the majority of the molecule and the DNA from this section codes for biochemical products related to providing energy to the cell. The other section of the mtDNA is called the control region and it is responsible for regulating the production of the gene products from the coding region. Within the control region there are two regions that have been found to contain a disproportionate number of variations in humans. These regions are called Hypervariable Region 1 and Hypervariable Region 2, or HV1 and HV2. HV1 is approximately 342 bp and HV2 is approximately 268 bp.
There are five major steps to mtDNA analysis. First, the sample is visually examined, cleaned, and prepared. Cleaning is extremely important because extraneous cells from handling can easily contaminate a sample. Usually the sample is immersed in detergent and an ultrasonic bath. Teeth and bones are sanded and cross-sectioned. In teeth, the dentin and pulp are used in the analysis. In all cases, the sample is ground to a powder and then placed in an extraction solution to release the cellular material, including the mtDNA, from the cells.
The second step involves extracting the mtDNA from the cellular material. This is accomplished by adding to the solution a mixture of chemicals that separate DNA from other organic molecules and then spinning the mixture in an ultracentrifuge. The mtDNA is concentrated in the top layer and then purified. The third step involves a technique called PCR, polymerase chain reaction, which uses carefully regulated cycles of heating and cooling to produce many copies of the mtDNA. This process is called amplification. After amplification, the mtDNA product is purified and quantified to ensure that the PCR yielded the expected quantity of mtDNA. The final step in mtDNA analysis is sequencing the amplified mtDNA. This is done using a technique similar to PCR, but special fluorescently labeled nucleotides that terminate the growth of a strand are added to the solution. This technique is referred to as Sanger's method and the result is many strands of DNA that vary in length by one nucleotide. This collection of DNA is then sorted by length, using a technique called gel electrophoresis. A fluorescence detector then reads the labels at the end of each strand of DNA and computer software reconstructs the mtDNA sequence. Finally, a DNA examiner edits and verifies the sequence.
When performing mitochondrial DNA analysis, about 610 bp are sequenced and compared to a standard. Any nucleotides in the sample sequence that differ from this standard are listed by location and nucleotide. For example, if a sample contained cytosine at position 263 while the standard contained adenine in this location, then the results would be presented 263 C.
The FBI has been using mtDNA to solve crimes since 1996. By 2002, they had processed more than 500 cases using mtDNA analysis and had established the National Missing Persons DNA Database to gather information on missing persons for the law enforcement community. A database of mitochondrial DNA can also be accessed through the FBI's CODIS (Combined DNA Index System) software. Mitochondrial DNA has been used successfully in a broad range of instances such as solving missing persons cases and identifying human remains and disaster victims. In 2005, the FBI decided to expand its mitochondrial DNA work and planned to open four new facilities focusing directly on mitochondrial DNA analysis.
SEE ALSO Human migration patterns; Identification of the son of Louis XVI and Marie Antoinette; Mitochondrial DNA typing.