The PCR amplification of the mitochondrial control region
There are 2 hypervariable regions within the control region of the mitochondria. This exercise amplifies just one of these. For more definitive results, both should be amplified and sequenced. This exercise will permit us to have a rough idea of the origins of our maternal line and we will be able to attribute ourselves to various tribes throughout the world. The human mitochondrial genome (genbank file).
Forward Primer5’-TTAACTCCACCATTAGCACC-3’
Reverse Primer5’-GAGGATGGTGGTCAAGGGAC-3’
PCR the previously extract DNA samples
Pour 2% agarose into casting apparatus in refrigerator
2 gels per class need to be made → 100ml of TBE with 2g agarose
add 5μl SYBR safe solution into the molten agarose before casting
place 2 sets of combs into the gel → at one end and in the middle
load gel with DNA ladder and PCR
Run gel at 120V for 20 minutes
Visualize on UV transilluminator
Document with camera to verify amplification
The instructor will submit the viable reactions for sequencing
In addition to the 23 chromosomes inherited from mother and 23 chromosomes inherited from father, humans have an additional genome that is only inherited from the mother. This genome comes from the endosymbiotic organelle, the mitochondrion.
Mitochondria are thought to have arisen in the eukaryotic line when bacteria capable of detoxifying the deadly effects of atmospheric oxygen were engulfed by a eukaryote that did not proceed to consume it. Over the course of time, these formerly free-living bacteria became dependent on the eukaryotic cell environment while providing the benefit to the host cell of aerobic respiration. Hallmarks of this endosymbiotic event include: the inner prokaryotic membrane surrounded by the outer eukaryotic membrane, the presence of prokaryotic ribosomes and most significantly, the circular prokaryotic chromosome. Mitochondria still replicate independently of the host cell but can not survive outside of this cellular environment. Animal mitochondria have the simplest genomes of all mitochondrial genomes, ranging from 11-28kb. The human mitochondrial genome consists of 37 genes which are almost all devoted to processing ATP through oxidative phosphorylation.
Human mitochondrial genome
The human mitochondrial genome (genbank file) consists of 16,569 nucleotides (16.6kb). While most of this 16.6kb genome consists of protein encoding genes, approximately 1.2kb non-coding DNA takes part in signals that control the expression of these genes and replication processes. It is the area of DNA where the double-strandedness is displaced and having the name D-loop (displacement loop). Mutations in this area generally have very little effect on the functioning of the mitochondria. Because of this reduced selection pressure on this area, this control region is also referred to as the hypervariable region. This hypervariable region actually has 10 times more SNPs than the nuclear genome. Due to this abundance of mutations, it is possible to track down the maternal line of an individual. Why just maternal? The human oocyte contains many mitochondria while sperm cells only contain mitochondria that power the flagellar motion. Upon fertilization, the flagellum and the associated mitochondria are lost, leaving the zygote with only maternal mitochondria.
The cluster of SNPs found in the mitochondrial control region are linked and are always inherited together. Because of the lack of paternal contribution, this linkage is referred to as a haplotype, or “half-type”. Tracking these polymorphic haplotypes, a family tree of humans was developed in the 1980s which concluded that humans arose from a metaphorical “Mitochondrial Eve” 200,000 years ago. As a metaphor to the Biblical Eve, this alludes to an origin but unlike the Biblical event, this does not mean that it was a single woman that gave rise to all of modern humanity. On the contrary, the metaphor merely indicates that a series of females; sisters and cousins, of this line gave rise to modern humans.
Migration map of mitochondrial haplogroups. Numbers represent 1000 years ago.
The use of mitochondria for this analysis provides great flexibility, especially from ancient sources. Unlike the nuclear genome which only has 2 copies of DNA per cell, the mitochondria are abundant in number and provide many copies of genome per cell. Ancient sources of DNA in fossils will most often have degradation of the DNA. The mitochondrial genome is just as likely to undergo degradation over time, however the high copy number allows for gaps to be filled in easily. SNPs do not alter the overall size of the hypervariable region, therefore amplification by PCR can not resolve these differences based on agarose gel migration. However, amplicons (amplified copies) can be sent for sequencing whereby each nucleotide can be called out in succession and reveal the specific SNPs.
Alu’s are unique SINEs that appear in the primate lineage and reveal the lineage and diversification of primates. While retrotransposons can disrupt gene (as in some cases of hemophilia), they often land outside of genes or within introns without effect. One example of a non-disruptive Alu element in humans is found in the location called PV92 on chromosome 16. This element is of the youngest subfamily of Alu, called Ya5.
Since PV92 does not cause any deleterious effects, it can be used as a non-selected marker to illustrate lineage. Some people have an Alu element int his location while others do not. The presence or absence of this marker is viewed as an allele. This lab uses primer that flank the location of the Alu insertion that span 416 bp. If an Alu is present, the amplified DNA will be 300bp larger (the size of an Alu) at 731bp.
Drawings dating to approximately 30,000 years ago in the Chauvet CaveWhat constitutes being human? Many will point at a cultural identity and leaving long-standing remnants of that culture. Such prehistorical artifacts like cave drawings and tools provide an anthropological framework for identifying what it is to be human, but the biological identity remains locked in the history of our DNA.
Spear points of the Clovis Culture in the Americas dating to approximately 13,000 years ago.
The Great Apes
Phylogenetic tree generated with Cytochrome Oxidase I (COI) genes.
Homo sapiens represent a branch of primates in the line of Great Apes. The family of Great Apes consists of four extant genera: Homo, Pan, Gorilla, Pongo. Karyotype analysis (Yunis et al., 1982) reveals a shared genomic structure between the Great Apes. While humans have 46 chromosomes, the other Great Apes have 48. Molecular evidence at the DNA level indicates that Human Chromosome 2 is a fusion of 2 individual chromosomes. In the other Great Apes, these 2 Chromosomes are referred to as 2p and 2q to illustrate their synteny to the human counterpart.
The Pan-Homo divergence. A display at the Cradle of Humankind illuminates the skulls of two extant Hominini with a series of model fossils from the Hominina subtribe of Austrolopithecina and Homo
Chimpanzees (Pan) are the closest living relatives to modern humans. It is commonly cited that less than 2% differences in their nucleotide sequences exist with humans (Chimpanzee Sequencing and Analysis Consortium, 2005). More recent findings in comparing the complement of genes (including duplication and gene loss events) now describes the difference in genomes at about 6% (Demuth JP, et al., 2006).
The Genus Homo
An underground lake at inside the Sterkfontein Cave system at the Cradle of Humankind (South Africa)
The rise of the human lineage is thought to arise in Africa. Fossils of Austroloptihs (southern apes) found in death traps, like those at the Cradle of Humankind, reveal a historical record of organisms inhabiting the landscape. The breaks in the ceiling of the caves provide opportunities for animals to fall inside these caves to their death. The limestone deposits of the caves serve as an environment for fossilization and mineralization of their remains. An abundance of fossilized hominids in these caves including Australopithecus africanus, Australopithecus prometheus, Paranthropus boisei, and the newly discovered Homo naledi continue to reveal the natural history of the genus Homo from 2.6 million to 200,000 years ago.
The entrance to the archaeological site at Sterkfontein, Cradle of Humankind (South Africa).
Ancient DNA of Humans
In 2008, a piece of a finger bone and a molar from a Siberian Cave were found that differed slightly from that of modern humans. The cave, called Denisova Cave, maintains an average temperature of 0ºC year round and was suspected to contain viable soft tissue. Bones in this cave were discovered that had similarities to modern humans and Neandertals. An initial mitochondrial DNA analysis revealed that these beings represented a distinct line of humans that overlapped with them in time (Krause et al., 2010). Analysis of the full nuclear genome followed and indicated that interbreeding existed between these Denisovans, Neandertals and modern humans (Reich et al., 2010). Furthermore, analysis of DNA from a 400,000 year old femur in Spain revealed that these three lines diverged from the species Homo heidelbergensis and that Denisovans were closest in sequence (Meyer et al., 2016).
Between modern humans, markers found in the mtDNA can be used to trace the migrations and origins along the maternal line. Similarly, VNTRs found on the Y chromosome have revealed migration patterns along paternal lines within men. Other markers, like the insertion points of transposable elements can be used to further describe the genetics and inheritance of modern humans while providing a snapshot into evolutionary history.
Yunis JJ, Prakash O. The origin of man: a chromosomal pictorial legacy. Science. 1982 Mar 19;215(4539):1525-30.
Chimpanzee Sequencing and Analysis Consortium. Initial sequence of the chimpanzee genome and comparison with the human genome. Nature. 2005 Sep 1;437(7055):69-87.
Krause, Johannes; Fu, Qiaomei; Good, Jeffrey M.; Viola, Bence; Shunkov, Michael V.; Derevianko, Anatoli P. & Pääbo, Svante (2010), “The complete mitochondrial DNA genome of an unknown hominin from southern Siberia”, Nature, 464 (7290): 894–897, doi:10.1038/nature08976, PMID 20336068
Reich, David; Green, Richard E.; Kircher, Martin; Krause, Johannes; Patterson, Nick; Durand, Eric Y.; Viola, Bence; Briggs, Adrian W. & Stenzel, Udo (2010), “Genetic history of an archaic hominin group from Denisova Cave in Siberia”, Nature, 468 (7327): 1053–1060, doi:10.1038/nature09710, PMID 21179161
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