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 non-

coding 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 self-

reported 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 Reference Mutation rCRS to Is it a known Haplogroup

Position Sequence Position your sequence polymorphism? Designations for
this site
250 16224 T to C Yes N/R: Subgroup Uk
260 16234 C to T Yes
337 16311 T to C Yes 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/simple-

tree-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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and revision of the Cambridge reference sequence for human mitochondrial DNA. Nature Genet.
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Finnila, S., Lehtonen, M.S., Majamaa, K. 2001. Phylogenetic Network for European mtDNA. Am. J. Hum.
Genet. 68: 1475–1484.

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. Hass, eds.)
Department of Biology, The Pennsylvania State University, University Park, PA.

Mitchell, S.L., Goodloe, R., Brown-Gentry, K., Pendergrass, S.A., Murdock, D.G., Crawford, D.C., 2014.
Characterization of mitochondrial haplogroups in a large population‑based sample from the
United States. Hum. Genet.

Stoneking, M. 2000. Hypervariable Sites in the mtDNA Control Region Are Mutational Hotspots. Am. J.
Hum. Genet. 69: 1029-1032.