The Analysis of Homo sapian Mitochondrial DNA in order to determine Maternal
Haplogroups
Introduction
Mitochondrial DNA exists as discrete, circular chromosomes of roughly 16 Kb within the
mitochondria of body cells. As the scientific understanding of gene replication and inheritance increases
through the use of new methods and technology, mitochondrial DNA is becoming a prominent material
used for understanding evolution, human distribution and maternally inherited disease patterns
(Mitchell et al., 2014). Mitochondrial DNA is particularly useful due to its unique heritability and
evolutionary characteristics. For instance, since mitochondrial DNA is found within the mitochondria and
human mitochondria are maternally inherited, mitochondrial DNA is only passed from mothers to
children and thus, can be traced exclusively along the maternal line (Mitchell et al., 2014). Additionally,
mitochondrial chromosomes exhibit a high mutation rate due to two distinct hypervariable regions that
experience frequent singular nucleotide base substitutions and undergo evolution rapidly. These noncoding regions, due to their relatively rapid evolution, exist in many different forms within the human
species (Stoneking, 2000). Through the collection and analysis of mitochondrial DNA from various
human samples, the common mutations seen within human samples can be sorted into haplogroups
that correspond to human geographic distribution over time. Previous data collections as well as the
understanding of mitochondrial maternal inheritance allow scientists to determine the geographic origin
of a sample’s maternal lineage through analysis and comparison of mitochondrial DNA.
In this experiment, the effectiveness of using human mitochondrial DNA to determine maternal
lineage is tested through the extraction, sequencing and analysis of a single human’s mitochondrial
DNA. The DNA, including the rapidly-evolving hypervariable regions, is extracted from a Homo sapien, or
human, cheek and subsequently isolated, amplified and sequenced to find a portion of the human’s
mitochondrial genome sequence. To test the effectiveness of mitochondrial DNA as an identifier of a
human’s maternal lineage and haplogroup, the collected sequence is then compared to a reference
sequence and known polymorphisms to determine the estimated maternal origin of the human. It is
expected that the singular nucleotide polymorphisms that exist within the human’s sequenced
mitochondrial DNA will match the haplogroup corresponding to her self-reported maternal origin.
Materials and Methods
The methods used for this experiment are outlined in the Human Genetic Variation Lab Manua
(Hass and Nelson, 2014)l. First, cells were swabbed from the inside of the human cheek and collected in
sterile containers to begin the extraction of mitochondrial DNA. The cells were subject to Qiagen DNeasy
Tissue Extraction where the mitochondrial DNA was separated and maintained while the cell tissues
were degraded by proteinase K. After the DNA was incubated, it was isolated via a series of buffer
additions and centrifuge cycles. After these cycles, the pure DNA sample was kept for subsequent
amplification while the other cellular materials were discarded. Next, the DNA was exponentially
amplified via the Polymerase Chain Reaction to increase the amount of usable DNA available for
sequencing. Since the isolated DNA concentration was low, at 18 ng/µl, 3 µl of the DNA solution was
added to 47 µl of the PCR mastermix in the experimental tubes to ensure that the PCR primers and Taq
polymerase had enough DNA to perform a successful amplification. To confirm that DNA samples
underwent a successful PCR procedure and enough DNA was produced, the amplified DNA was dyed
and subject to gel electrophoresis alongside a single DNA ladder and a negative control. Since the DNA
did not show up as distinct bands on the electrophoresis gel (i.e. the experimental lanes appeared
empty), it was assumed that the DNA existed in low concentrations and both experimental DNA samples
had to be combined to ensure successful sequencing. The combined DNA samples were then purified via
another series of buffer additions and centrifugations to prepare them for sequencing. The samples
were then sequenced and uploaded onto computer files to be analyzed. In the analysis, the collected
sequences were compared to the rCRS, or revised Cambrian Reference Sequence, where single
nucleotide polymorphisms were identified. These polymorphisms found within the sample were then
cross-referenced to existing mitochondrial DNA polymorphisms. By comparison to previously
determined polymorphisms, the haplogroup of the human was determined and compared to her selfreported maternal origin.
Results and Discussion
After collection and sequencing, the DNA collected from human cheek cells was able to be
analyzed in a variety of ways. In order to determine whether or not a significant amount of DNA was
present, the first analysis done on the sample was gel electrophoresis. Figure 1 shows an image of the
electrophoresis gel taken while it was being exposed to UV light. Even though both the DNA samples
and the ladder were stained with ethidium bromide, a stain that fluoresces under UV light, only the
ladder appeared on the gel when exposed to ultraviolet radiation. Lanes 2 and 3, the sample DNA lanes,
appeared empty. This indicates that the DNA existed in small enough quantities that it could not be
viewed within the agarose gel. Since the electrophoresis process separates the DNA based upon the size
of the DNA fragment, with the largest fragments traveling the shortest distance, if visible, the
mitochondrial sample DNA would have appeared in line with the DNA ladder wrung that corresponded
to close to 16 kb. When analyzing the gel in figure 1, it is important to note that the negative control
lane (lane 4) also appeared empty. This indicates that there was no extraneous DNA in the PCR
mastermix during the PCR process which ensures that the only DNA that was amplified and analyzed
was the DNA isolated from the human cells.
Figure 1. Electrophoresis gel of amplified human cheek cell mitochondrial DNA.
After combining the DNA samples to ensure that the mitochondrial DNA would be able to be
further analyzed, the DNA was purified once more to guarantee a clear sequence. Upon viewing the
DNA sequence with the Mega software, the peaks were distinguishable after the 21st position on the
sequence. Prior to that position, a significant amount of noise prevented the software from identifying
which nucleotide existed at specific designations. This is most likely due to the fact that the PCR primer
was meant to bind the DNA strand and amplify after position 22. From position 22 to position 417, the
peaks contained very little noise and the individual base pairs were easily identified. The peaks, though,
did appear small, most likely due to the low concentrations of DNA that were analyzed. After the
sequence’s peaks were viewed and checked for noise, the determined sequence was compared to the
rCRS to determine which single nucleotide polymorphisms were present within the sample. After the
deletion of any N’s, or undetermined nucleotides, within the sequence, position 22 on the sample
sequence was aligned with position 15996 on the rCRS and the two sequences were checked for single
base pair differences. Figure 2 indicates that the Human sample and the rCRS differed by three single
nucleotide differences. These differences were then compared to the common polymorphisms exhibited
by different Human mitochondrial DNA haplogroups.
Alignment
Position
Reference
Sequence Position
Mutation rCRS to
your sequence
Is it a known
polymorphism?
250
260
337
16224
16234
16311
T to C
C to T
T to C
Yes
Yes
Yes
Haplogroup
Designations for
this site
N/R: Subgroup Uk
M: Subgroup Q;
N/R: Subgroup
H/V, Uk
Figure 2. Chart indicating the differences between the Human sample sequence and the rCRS and the
significance of those differences in regards to haplotypes.
All three of the differences exhibited by the Human sample were recognized as known
polymorphisms. Two of the three nucleotide substitutions corresponded to known haplogroups listed
on the mitomap chart. (found at http://www.mitomap.org/pub/MITOMAP/MitomapFigures/simpletree-mitomap- 2012.pdf ) For alignment position 250, or 16224 on the rCRS, the only haplogroup that
exhibited the thymine to cytosine mutation was the N/R: subgroup Uk. This same haplogroup was listed
as a group that exhibited the thymine to cytosine mutation at position 16311 on the rCRS, another
mutation that the human sample sequence indicated. Even though this sample sequence represents
only a small portion of the mitochondrial DNA, the sequence indicates that human who donated this
sequence belongs to the N/R: Subgroup Uk. According to Finnila and Majamaa ,the four most common
Haplolgroups seen in those of European heritage are HV, UK, TJ and WIX. The differences between these
haplogroups, and most other haplogroups, appear primarily in the variable regions and not in the
structural, tRNA or rRNA regions of the mitochondrial DNA (2001). The determination that the Uk
haplogroup indicates European origin coincides with the self-reported maternal heritage from the of the
human who donated the sample. The self-reported maternal lineage of the human includes Poland and
Lithuania, both European countries. According to the information provided by both self-report and the
sample sequence’s polymorphisms, the analysis of mitochondrial DNA was successful in identifying the
maternal lineage of the human subject.
After being used to determine the haplogroup of the sample, the sequence was subject to a
BLAST search to find the gene and species to which it corresponds most. The BLAST search indicated
that the sequence was 99 percent identical to the Homo sapien mitochondrion complete genome,
confirming that the sequence is both human and from the mitochondria. The sequence exhibited only
three nucleotide differences from the closest match sequence. According to the publication
corresponding to the human mitochondrion genome sequence, the listed sequence is actually the same
rCRS that was used during the Mega sequence comparison. The original CRS existed first as a sample
from a sample of European human mitochiondrial DNA with some bovine and HeLa sequences included.
Later findings determined that the original CRS, which came from a single human sample, contained
both errors and rare mutations that made it inaccurate as a comparison sequence for other
mitochondrial DNA. Thus, the sequence was then modified into the rCRS sequence that was used for
comparison in this experiment and listed as the first matching sequence found by the BLAST search
(Andres et al., 1999). Since the searched sequence is the rCRS, the three nucleotide differences between
the sample sequence and the search sequence coincide with the three polymorphisms seen between
the rCRS and the sample sequence in the Mega comparison. The nucleotide differences are simply single
base nucleotide substitutions which are point mutations that do not result in any frameshifts. The BLAST
search further confirmed that the analyzed sequence was successfully extracted from human cells and
isolated from mitochondria.
This experiment proved that extraction of cheek cells from a human sample and subsequent
isolation and amplification of the mitochondrial DNA allows for sequencing and analysis of the
aforementioned maternally inherited DNA. The DNA was easily isolated and, upon sequencing, showed
little noise which allowed for the nucleotide order to be confidently determined. This sequence could
then be compared to a standard reference mitochondrial sequence and searched for common
polymorphisms. With accuracy, the single nucleotide differences seen in the sample sequence could be
traced to the reported maternal haplogroup of the human who donated the sample. This simple yet
accurate process could be useful in many areas of science, including forensics and medicine. In forensics,
DNA found in a crime scene could allow scientists to eliminate suspects by comparing the
polymorphisms on the mitochondrial DNA of the crime scene sample to those of the subjects. In
medicine, the mitochondrial DNA may help in the understanding of maternally inherited diseases and
disorders (Mitchell et al., 2014). Overall, mitochondrial DNA, due to its specific characteristics and
relatively easy analysis, is a piece of genetic material that is useful in many areas of scientific study.
Literature Cited
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Hass, C.A., and K. Nelson. 2014. A molecular investigation of human genetic variation. In A Laboratory
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