Jackie Krevitz

A molecular investigation in human genetic variation

Biol 220M
TA: Drew Fister
3/24/14
Introduction:
Within human populations, much variation occurs within DNA sequences that allows us
to tell humans apart based on their DNA. Specifically, in the hypervariable regions HV1 and
HV2 of the mitochondrial genome, much variation occurs between humans (Biol 220M Lab
Manual,2014). Humans have both nuclear DNA, found in the nucleus, and mitochondrial DNA,
found in mitochondria (Biol 220M Lab Manual 2014). Mitochondrial DNA contains more than
15,000 base pairs (Steffens 2014). The mitochondrial DNA, or mtDNA, contains 11 genes for
coding proteins, two genes for rRNA, 22 genes for tRNA, and the control region (Biol 220M Lab
Manual 2014). The control region contains the hypervariable regions 1 and 2, which are the
most variable parts of mitochondrial DNA (Steffens 2014).
Mitochondria, and specifically their DNA, provide much insight into the history of
human and geographic distribution of humans. Mitochondria are always passed on from the
mother, so tracing the HV1 and HV2 region throughout a region can provide information on
human lineages and history (Biol 220M Lab Manual 2014). HV1 and HV2 are each about 250250 base pairs long and vary greatly (Steffens 2014). Variations in mitochondrial DNA not only
allow researchers to trace humans through time, but also can have phenotypic effects, including
causing a variation in endurance capacity (Biol 220M Lab Manual 2014). As well, mutations in
mitochondrial DNA have been associated with breast, colorectal, and lung cancer cells (Biol
220M Lab Manual 2014).
Different mutations at certain sites in mitochondrial DNA can indicate where that lineage
originated. These differences in mtDNA have been characterized into haplogroups A-Z based on
time of discovery and geography (Biol 220M Lab Manual 2014). Certain haplotypes can be
traced back to different regions of the world to help determine the migration patterns of ancestral

populations over time (Biol 220M Lab Manual 2014). The mitochondrial most recent common
ancestor, or mt-MRCA, can be traced back thousands of years to the single maternal lineage
from which all modern humans have descended (Biol 220M Lab Manual 2014).
The analysis of mitochondrial DNA can be used for reasons besides the high variability
of HV1 and HV2. Human cells often contain hundreds of copies of mitochondrial DNA. When
using PCR amplification, the increased number of copies of mitochondrial DNA allows for easier
amplification than when nuclear DNA is used (Biol 220M Lab Manual 2014).
Sequencing the HV1 region alone does not provide any meaningful results without the
comparison to a reference sequence. Two main reference sequences have historically been used
to analyze and draw conclusions from mitochondrial DNA. The Cambridge Reference Sequence
was originally isolated in 1981 and has been used for many years (Biol 220M Lab Manual 2014).
The Reconstructed Sapiens Reference Sequence, or RSRS, was determined using all of the
available mitochondrial genomes from Homo neanderthalensis (Biol 220M Lab Manual 2014).
This sequence was proposed to be put into use in 2012 by Behar et al, but controversy over
which sequence should be used continues (Biol 220M Lab Manual 2014). In todays scientific
world, the revised Cambridge Reference Sequence is most often used when comparing DNA
sequences.
The purpose of this experiment, according to the Biology 220M lab manual, was to
attempt to identify the haplotype group of the HV1 region of human mitochondrial DNA. This
was done by extracting a DNA sample, amplifying the HV1 region of mitochondrial DNA using
PCR, examining PCR products using gel electrophoresis, sequencing the DNA sample, and
analyzing the results obtained (Biol 220M Lab Manual 2014).
Materials and Methods:
DNA was collected and isolated from the subjects inner cheek as per the lab manual.
DNA was extracted using proper technique and amplified using HV1 PCR amplification of 49
microliters of PCR Master cocktail and 1 microliter of sample DNA. Gel Electrophoresis was

done on PCR products and purification of amplified DNA was carried out. The sequencing plate
was prepared and DNA was sequenced. Analysis of DNA sequence compared to the reference
sequence given was carried out and recorded. All procedures were followed as per the lab
manual; however, a few issues arose. The isolated DNA could not be sequenced, and the TA
provided a replacement sequence. All other procedures were carried out as expected. (Biol
220M Lab Manual 2014).
Results and Discussion:
PCR and Gel Electrophoresis:
After PCR amplification, a gel electrophoresis was carried out to ensure the PCR
amplification was successful. The amplified region was very difficult to see in the gel
electrophoresis, as seen in figure 1 below.

Although difficult to distinguish, the first row, row

ladder, used

Figure 1: Gel electrophoresis

The next two, rows 2 and 3, contained my DNA. The last

5
4
3
2
1

1, is the DNA
for comparison.
two, rows 4 and

5, contained the DNA of Corinne Renner. The dim nature of the gel indicated that PCR
amplification was not as successful as hoped, but DNA was sent to be sequenced. However,
when the plate returned, the DNA sequence was seen as messy and could not be used. A
replacement sequence was given to allow for the completion of this lab analysis.
A. Sequence Data:
Possibly due to the fact that it was the beginning of the data, noise was observed from the
beginning of the sequence at base 34 until base 54. However, the underlying trace still indicates
the proper bases, as shown in figure 2 on the following page:

Figure 2: Trace of sample mitochondrial DNA

At base 90, noise is observed as seen in figure 3. However, the underlying trace still indicates
the proper base pairs.

Figure 3: Trace of sample mitochondrial DNA

The rest of the trace is as expected and matches the base pairs seen.
B/C. Sequence Analysis and Haplotype determinations:
During data analysis, 33 bases were deleted from the beginning of the DNA sequence.
After shifting, the data matched with the given reference sequences starting at base pair 49. The
3 end of the L primer in my sequence, found at base pair 22, was T. A portion of the sequence
comparison can be seen in figure 4 below.

Figure 4: Comparison of sample mitochondrial DNA

The deviations from the reference sequence, along with haplotype designations can be found in
Table 1 on the following page.
Table 1: CAH HV1 Sequence Analysis and Deviation from reference sequence

Alignment
Position

Reference
Sequence
Position

Mutation
rCRS to your
sequence

Is this a known
polymorphism?

Haplogroup
Designations for
this site (tentative
indicated
haplotype in
bold)

250

16224

T to C

Yes

285
337

16259
16331

C to T
T to C

Yes
Yes

N/R: Subgroup
Uk
none
none

The only haplotype found was subgroup Uk, but those results are for a different mutation at the
same site. Uk is a haplogroup found mostly in east Europe and within Caucasus, but also in
Africa, the Middle East, and Central Asia (O. Derbeneva). Because this DNA sample was
assigned by a TA, and was not actually my mitochondrial DNA, it was impossible to determine if
these estimated haplogroups were consistent with my family history.
In A Mitochondrial Etiology of Neurodegenerative Diseases: Evidence from Parkinsons
Disease, the effects of mutations in mitochondrial DNA were observed, specifically relating to
Parkinsons disease (PD). Much research has been done regarding PD and mitochondrial
mutations, specifically regarding different haplogroups. They found that a mutation within
haplogroup H can increase the risk of PD, but mutations within haplogroup Uk can actually
decrease the risk of PD (Khusnutdinova 2014). Additionally, people with both haplogroup Uk
and H are more likely to have additional mutations in their mitochondrial DNA that may
contribute to the risk of PD (Khusnutdinova 2014). More research needs to be done to determine
the actual effects of such mutations.
D. Blast search:
For the first result returned from the blast search, the percent identity was 99% to the
Homo sapiens mitochondrion, complete genome. There were 3 differences between my
sequence and the closest match, including a C for T single base pair substitution at base #202, a
single base pair substitution of T for C at base #237, and a single base pair substitution of C for T
at base #279.
The article, Sequence and organization of the human mitochondrial genome (1981),
contains the closest match to my sequenced mitochondrial DNA. In this article, the researchers
completely sequenced human mitochondrial DNA. In addition, many specific genes for coding

proteins were predicted to have been found. They also discovered that there are some inactive,
non-coding bases within mitochondrial DNA and that STOP codons are not included within
the mitochondrial DNA (Anderson 1981).

References:
Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H., Coulson, A.R., Drouin, J., Eperon,
I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J., Staden, R. and
Young, I.G. Sequence and organization of the human mitochondrial genome. Nature 290
(5806), 457-465 (1981)
Derbeneva, O. HaplogroupMarkers [Internet]. Mitomap2009 [cited 2014 March 22]. Available
from http://www.mitomap.org/bin/view.pl/MITOMAP/HaplogroupMarkers
Khusnutdinova, E, I. Gilyazova, E. Ruiz-Pesini, O. Derbeneva, R. Khusainova, I. Khidiyatova,
R. Magzhanov, and D.C. Wallace. "A Mitochondrial Etiology of Neurodegenerative
Diseases: Evidence from Parkinson's Disease." Annals of the New York Academy of
Sciences 1147 (2008): 1-20. Web. 22 Mar. 2014.
Steffens, D.L., and R. Roy. "Sequence Analysis of Mitochondrial DNA Hypervariable Regions
Using Infrared Fluorescence Detection." BioTechniques 24.6 (1998): 1044-046. Web. 21
Mar. 2014.