Forks in the Evolutionary Road: Tracing Genetic
Variation in the Hypervariable Regions of Human
Mitochondrial DNA
Victoria Cavrich
TA: Drew Fister
BIO 220M
Section:001
3/24/2014
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Introduction:
About two hundred thousand years ago, the Earth began to see the rise of the species that
would come to dominate the planet and shape it into the world as it is today. That species was of
course, Homo sapiens, or as they are more commonly known, human beings. Humans are
believed to have originated in the eastern regions of Africa. (O’Neill) The species slowly began
to migrate to different areas of the continent and eventually spread to the rest of the world as
well. By examining ancient fossils and artifacts, evidence of human migration can be found by
comparing skeletal structure of bones and skulls found in different locations or identifying the
ages of these remains through carbon dating. However, the migration of Homo sapiens across
the Earth can be traced on a much more miniscule level. By examining the mitochondrial DNA
in Homo sapiens from both today and the distant past, one can trace the multitude of divergences
from the genetic material inherited from the lineage of ancestral humans.
Mitochondria are, at their most basic form, the organelles in the cell that provide the
energy necessary for an organism to function. Mitochondria are unique in that they are the only
organelles, other than the chloroplasts found in plant cells, which have their own genome that is
separate from the genetic material found in the nucleus of the cell. This genome is circular and
contains about 16,500 base pairs. (Genetics Home Reference) These genes can code for proteins,
rRNA, and tRNA. Additionally, mitochondrial DNA contains a specific non-coding control
region that is the most variable portion of the DNA due to two hypervariable regions known as
HV1 and HV2. Mutations in these regions can lead to either positive effects like increased
endurance or can be associated with certain diseases like cancer, diabetes, deafness, and other
various syndromes and disorders.
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The other unique characterization of mitochondrial DNA is that it is not inherited in the
same fashion as all of the other genetic information in the human genome is typically passed
down. Mitochondrial DNA is passed down through maternal genes, meaning that only the
mother’s genetic material from the mitochondria found in the fertilized egg cell is passed to her
offspring. (National Genetics and Genomics Education Center) This pattern of inheritance
allows geneticists to trace diseases and other conditions caused by mutations in the mitochondria
through family trees. These mutations can be traced back through multiple generations by
sequencing the mitochondrial DNA of the mothers with affected children to determine where the
mutation originated and was eventually transferred to the offspring.
Tracing mitochondrial DNA in family lines can be expanded to a much larger scale.
Applying the same principle that mitochondrial DNA only passes from the mother to her
offspring, the lineages of entire subgroups of humans can be mapped out. People can begin to
trace their origins back to ancient times when humans were first beginning to make the Earth
their own. The mutations found in the HV1 and HV2 regions of the mitochondrial DNA serve as
markers, showing where certain subgroups of Homo sapiens migrated to and from throughout the
course of human history.
In order to begin tracing the mutations found in mitochondrial DNA, several processes
are used to generate the samples necessary for sequencing. The samples of DNA must first be
purified and processed by adding certain enzymes and buffers and putting the samples into a
centrifuge. Polymerase Chain Reaction, or PCR for short, is then used to generate huge amounts
of DNA by heating the original samples of DNA until they denature and then replicating those
samples with the primers and nucleotides found in the PCR mixes. The replicated samples are
then directly sequenced, or they can be run through the process of gel electrophoresis in which
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the samples are coated with special dyes that fluoresce under UV light and allows the samples to
be compared to DNA of known base pair length. The negative source of energy in the
electrophoresis machine pushes the DNA, which has a slightly negative charge, through the gel.
The further a fragment of DNA travels the smaller amount of base pairs it contains. In the
following experiments, samples of students’ DNA were obtained and put through these processes
in order to trace the origins of the mitochondrial DNA inherited by each student.
Materials and Methods:
Samples of the DNA were first obtained by swabbing the insides of the students’ mouths
with cotton swabs to collect cells. The swabs were then deposited in a microcentrifuge tube and
180 µl of Buffer ATL and 20 µl proteinase K in order to purify the raw DNA sample by
digesting any contaminant proteins. The samples were then allowed to incubate at 56° C for five
days. After the incubation period, the samples were removed and 200 µl of Buffer AL and 200
µl of ethanol were added to the tube. The samples were spun in a small centrifuge and then
transferred to a DNeasy Mini Spin Column. The column was spun at 8000 rpm for one minute
and the collection tube was discarded. 500 µl of Buffer AW1 was added to the sample and the
column was spun again at 8000 rpm. After the collection tube was discarded again, 500 µl of
Buffer AW2 was added and the column was then spun at 14,000 rpm for one minute. Finally 50
µl of Buffer AE was added to the spin column, the tube was allowed to incubate for one minute,
and the apparatus was spun at 8000 rpm once again. The sample of DNA was then put through a
NanoVue Spectrophotometer to determine amount of DNA present in the sample.
The samples of DNA were then added to the PCR Master cocktail, using 2 µl of the
sample DNA and 48 µl of the PCR mix. The samples were allowed to go through the PCR
process and were then either set aside to be put through the sequencing process or subjected to
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gel electrophoresis. The gel used in the electrophoresis process contained a DNA ladder
containing fragments of known base pair length and two samples of two different student’s
DNA. The gel electrophoresis process was carried out to completion and the gel was placed
under a UV light in order to allow the DNA to be seen.
Results and Discussion:
After the original sample of DNA had been purified, a small sample was tested with the
NanoVue Spectrophotometer to determine the amount of DNA in the sample. The sample was
determined to contain 58.5 ng/ µl of DNA. This was a medium amount of DNA, meaning that 2
µl of the sample would be necessary for the PCR process.
After the samples were put through the PCR process they were put through the gel
electrophoresis process. However, despite the relatively high amount of DNA present in the
samples, both students’ samples failed to appear under the UV light. This failure to appear was
not due the failure of the dye to adhere to the DNA as the ladder did appear under the UV light.
There is a possibility that the DNA had not been cut into small enough fragments that would
have been able to push through the gel. Another possibility is that not enough DNA from the
original sample had been deposited in the wells of the gel and as a result, could not be seen when
placed under the light.
Ladder DG1 DG2 VC1 VC2
Fig. 1 Gel Electrophoresis of Students’ Samples and Ladder
Legend:
Ladder- DNA ladder of known bp
length
DG- Dominic Gallanti Sample
VC- Victoria Cavrich Sample
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In addition to the failure of the DNA samples to appear during the gel electrophoresis
process, the sequencing process was not completed successfully as well. The resulting sequence
was too messy to be analyzed accurately and as a result, another sample sequence had to be used
in order to trace the examples of mutations in the mitochondrial DNA. This sequence was
simply known as Sequence Three. A cursory analysis of the sequence showed unidentifiable
base pairs for most of the first forty pairs due to “noise”. However, beyond this point all of the
exact base pairs were able to be sequenced up to about four hundred and nine base pairs.
The DNA sequence was then compared to a reference sequence provided by the
instructor. All the differences between the base pairs from the DNA sequence and the reference
sequence were noted. (Fig 2.) These sites were then researched to see if the mutation of the base
pairs was a common polymorphism. Once the mutation was confirmed to be a known
polymorphism, the haplogroup designation for the site was determined according to the site
where the mutation occurred.
Fig 2. DNA Sample and Reference Sequence Alignment
Alignment
Position
Reference
Sequence
Position
Mutation rCRS
to My Sequence
Known
Polymorphism?
29
33-34
35
36-39
42
127
151
360
419
16004
16008-16009
16011
16011-16014
16017
16082
16106
16315
16374
Noise
Noise
A to G
Noise
Noise
G to A
G to A
T to C
A Insertion
No
No
Yes
No
No
No
Yes
No
No
Haplogroup
Designation for
this Site
(tentative
indicated
haplogroup types
in bold)
N/a
N/a
N, M, L
N/a
N/a
N/a
J&T
N/a
N/a
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Two sites in the gene sequence were known polymorphisms. At site 16011, the A
nucleotide changed to a G nucleotide. This mutation did not have a specific haplogroup
designation and could be applied to the M, N, and L haplogroups. Similarly the mutation at site
16106 from a G to an A did not have a specific site listed for the haplogroup, but could be
averaged between the sites where mutations correspond to the J and T haplogroups. Since there
was an indication of the mutations typical of the J and T haplogroups, it seems logical that the
first known polymorphism corresponded to the N haplogroup since these two groups are
believed to have originally evolved from the N haplogroup. (Mitoweb) These haplogroups were
believed to have migrated from Africa into Eastern Europe thousands of years ago, indicating
that the subject that this DNA sample came from is most likely of Eastern European descent.
Performing a BLAST search on the DNA sequence resulted in a 99% match with the
complete genome of the mitochondrion from a Homo sapien. There were two differences
between the mitochondrion genome and the DNA sequence from the experiment. Both
differences were point mutations with one alteration from A to G and one alteration from C to T.
The genome used to compare the DNA sequence sample came from the experimentation of
several researchers attempts to revise the Cambridge reference sequence of mitochondrial DNA.
(Andrews) The researchers sequenced the HeLa mitochondrial DNA in its entirety from an
African source of origin. They found that there was .07% error found in the current CRS and
suggested that a revised version be used as a standard in research of mitochondrial DNA
lineages.
Further research of the migration of these haplogroups has been performed to determine
the connections between the inheritance of multiple sclerosis and the mtDNA haplogroup J. The
researchers collected around 248 samples of mitochondrial DNA from the patients’ blood and
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screened them for the presence of different haplogroups. The patients that the samples were
taken from ranged from having no symptoms of MS to a long family history of the disease.
They found that 25% of the patients with MS had the BM haplogroups, 20% had the J
haplogroup, about 13% had the K haplogroup, and about 3% had the M haplogroup. These
results led the researchers to believe that there was a link between the J and K haplogroups and
MS in Caucasian individuals. Additionally, they believed that the haplogroup J was a risk factor
for MS in Persian Caucasian populations. The researchers suggested carrying out follow-up
research in the connections between MS and LHON mutational disease since the optic nerve
atrophy was seen more frequently in MS patients with the J haplogroup.
There are still several areas of research and experimentation concerning the study of
mitochondrial and other types of human DNA. This procedure could be repeated in order to
obtain an actual sample that would sequence correctly instead of using a nonphysical sample
DNA sequence. Similarly, the experiment could be repeated using sources of DNA other than
saliva. Nucleic DNA samples could also be sequenced for comparison with the human genome
reference sequences being generated by the world’s top scientists. The full extent of the
information concerning the human genome and its mechanisms and inheritance is still unknown,
but further experimentation like this can serve to unlock some of the mysteries that still remain.
References:
Andrews, R. M., Kubacka, I., Chinnery, P. F., Lightowlers, R. N., Turnbull, D. M., & Howell, N.
(1999). Reanalysis and revision of the Cambridge reference sequence for human
mitochondrial DNA. Nature Genetics, 23(2), 147.
Hass, C.A., and K. Nelson. 2014. A molecular investigation of human genetic variation. In A
Laboratory Manual for Biology 220W: Populations and Communities. (Burpee, D. and C.
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Hass, eds.) Department of Biology, The Pennsylvania State University, University Park,
PA.
Houshmand, M., Sanati, M., Babrzadeh, F., Ardalan, A., Teimori, M., Vakilian, M., et al. (2005).
Population screening for association of mitochondrial haplogroups BM, J, K and M with
multiple sclerosis: interrelation between haplogroup J and MS in Persian patients.
Multiple Sclerosis, 11(6), 728-730. Retrieved March 19, 2014, from the PubMed
database.
Human mtDNA Migrations. (n.d.). Mitoweb. Retrieved March 19, 2014, from
http://www.mitomap.org/pub/MITOMAP/MitomapFigures
Mitochondrial DNA. (2014, March 17). Genetics Home Reference. Retrieved March 19, 2014,
from http://ghr.nlm.nih.gov/mitochondrial-dna.
Mitochondrial inheritance. (n.d.). National Genetics and Genomics Education Center. Retrieved
March 19, 2014, from http://www.geneticseducation.nhs.uk/genetics-glossary/212mitochondrial-inheritance.
Simplified mtDNA Lineages. (n.d.). Mitoweb. Retrieved March 19, 2014, from
http://www.mitomap.org/pub/MITOMAP/MitomapFigures/simple-tree-mitomap2012.pdf
O'Neil, D. (n.d.). Evolution of Modern Humans: Early Modern Homo sapiens. Anthropology
Tutorials. Retrieved March 19, 2014, from http://anthro.palomar.edu/homo2/mod_homo.