MOYAN JIA

MKJ5122

SECTION012

Human Genetic Variation: Modern
Homo Sapiens Haplogroup
Classification
BIOL 220W LAB REPORT

TA: Wilfried Guiblet

Partner: Xin Tang

2016/02/28

BIOL 220W

Moyan Jia

mkj5122

Human Genetic Variation: Modern Homo Sapiens Haplogroup Classification
1.0 Introduction
Homo Sapiens have the long history of evolution. With the general skim of whole
phenogenetic history, human evoke from primates and gain the traits of large brain
volume, bipedalism, and the ability of tool use. Within the group of modern
human, people are separated on different branches, i.e. haplogroups, of the
phenogenetic tree. Haplogroup is a genetic population group in which human
share same ancestor on maternal or paternal lineage [1]. How should modern
human be classified and distinguished is a complicated issue. The purpose of this
experiment is to analyze the relationship between modern human, as the
experimenter, and ancestor and to test what type of variation exists in genetic
scale.
Genetic information reveals the nature of haplogroup linkage. There are two
genomes inside the human body, the nuclear genome, known as the general
chromosomes, and the mitochondrial genome, which is a maternal, cyclic DNA
segment [2]. Mitochondrial DNA is bacteria originated, including 155569 bp
DNA, protein coding genes, rRNA genes, tRNA genes, and a non-coding control
region. The control region is highly variable, especially the hypervariable regions
HV1 and HV2 [3]. The mitochondrial DNA is widely used in phenogenetic
haplogroup distribution, which provides universal information about how an
individual is descended from maternal lineage [1].
For the purpose of comparison, a references sequence commonly known as rCRS,
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the revised Cambridge Reference Sequence, retrieved from Anderdson, 1981, is as
a sample of contrast and analysis. rCRS is not a real human genome discovered
early, it is rather a revised human genome modified for the purpose of genetic
engineering [4].
In this experiment, the experimenter’s DNA mkj5122_L15996_G5 is retrieved
from oral epithelium, processed with Polymerase Chain Reaction and gel
electrophoresis to duplicate and amplify DNA. The experimental DNA mkj5122 is
compared with rCRS. The certain known mutations are used to classify
haplogroup designation.
Hypothesis of this experiment is that the experimenter’s modern genome has high
similarity with rCRS; since the experimenter and all of her ancestors were born in
China, the experimenter’s DNA sequence has significant possibility to be
classified in M: Subgroup Q which originated in Asia [5].
2.0 Materials and Methods
To proceed this experiment, epidermal cells were collected inside cheek with
cotton swab. The cotton swab carrying with experimenter’s DNA was treated with
buffer and proteinase. Centrifuge and relative solutions were used to extract DNA;
PCR machine was used to analyze the DNA data; and sterile water was kept as the
control group [3].
200 microliter Buffer AL was added into mkj5122 samples and 200 microliter of
high concentration ethanol was added into DNA column to wash. 500 microliter
Buffer AW1, 500 microliter AW2, and 50 microliter AE were added into column
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companioned with centrifuge. 2 samples of experimenter mkj5122’s DNA, 2
samples of partner xzt5037’s DNA, and 1 sample of sterile water were
manipulated gel electrophoresis. After the cell lysis, binding, washing, and elution
processes, DNA samples were measured concentrations through the NanoVue
Spectrophotometer and determined the quantity of master cocktail added.
# DNA concentration
Amount of mater cocktail
>100 ng/µl
1 ng/µl
50-99 ng/µl
2 ng/µl
<10-49ng/µl
3 ng/µl
Table 1: Reference DNA Concentrations [3]
The concentration of extracted DNA samples were > 100ng/ml, so 1 microliter of
master cocktail was added.
The DNA was analyzed by PCR. Agarose was placed into a flask and mixed with
1X TAE buffer. The flask was heated for 35sec in microwave. Then the gel was
put at room temperature and cooled for 2 minutes. In order to view DNA ladder
under UV light, ethidium bromide dye was added into molten agarose. When
casting the gel, agarose should harden for 10min. Then the DNA samples were
loaded into wells. The gel electrophoresis well and samples match are recorded
into the table below.
Well
1
2
3
4
5
6
7

Sample
DNA ladder
Moyan Jia’s sample 1 mkj5122
Moyan Jia’s sample 2 mkj5122
XinTang’s sample 1 xzt5037
XinTang’s sample 2 xzt5037
Negative control
DNA ladder
Table 2: Wells and Samples Match
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The DNA result was then analyzed and compared with rCRS via MEGA.
3.0 Results
The gel electrophoresis result demonstrates that all the DNA segments and
negative control are on the same horizontal plate (shown in figure 1), which
indicates the result is not precious. Most experimenters’ data shows that gels
added master mix displaying non-sense results. The DNA purification was
operated again.

1

7

2 3 4 5 6

Figure 1: Gel Electrophoresis under UV, all segments ran same distance

After the DNA sequence was analyzed into specific nucleotides, experimenter
mkj5122’s DNA was compared and contrasted with rCRS. MEGA displays and
traces DNA in wave graphs (figure 2). All noisy and non-sense DNAs were
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ignored. The significant peaks were recorded and further analyzed.

Figure 2: Experimenter mkj5122’s DNA Trace
The mkj5122 sequence and rCRS were exported into alignment (figure 3), which
share high similarities. The mutated positions are not marked asterisks on top.
Eliminating portions with low confidence interval, i.e. the noisy segments, most
mutated positions are listed in table 3.

DNA Alignment from site #49 to #129

DNA Alignment from site #130 to #213

DNA Alignment from site #214 to #297
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Figure 3: Experimenter mkj5122’s DNA and rCRS Alignment
In order to transfer mitochondrial DNA alignment position to reference sequence
position, a formula was adapted:
# experimenter’s DNA – 22 +15996 = # reference sequence
# experimenter’s DNA

# rCRS

Mutation

Known Exist

166

16140

T to C

Yes[6]

209

16183

A to C

Yes[7]

215

16189

T to C

Yes[8]

220

16194

A to C

Yes[9]

222

16196

Inserted T

No

223

16197

C to T

Yes

227

16201

C to A

No

231

16205

C to A

No

240

16214

C to A

Yes

258

16232

C to A

Yes

260

16234

C to A

Yes

262

16236

C to A

No

265

16239

C to A

Yes

269

16243

T to C

Yes

284

16258

A to C

Yes

308

16282

C to A

Yes

319

16293

A to C

Yes

Table 3: The Mutation in experiment’s DNA from rCRS
The DNA sites marked “Yes” in “Known Exist” column in table 3 are determined
as known polymorphism. These site transitions are referred to haplogroup
determination. According to haplogroup markers, at sites 16183 and 16189 the
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mutations are consistent with N/R subgroup B [10].
4.0 Discussion
There are only 2 variations in experimenter mkj5122’s DNA that can be
determined as haplogroup markers in N/R subgroup B.

Figure 4: Simplified mtDNA lineages [11]
The experimenter and all relatives were born in China, so the DNA is supposed to be
found in Asia lineage. According to figure 4, N/R subgroup B originated from Africa,
migrated to Europe, and finally settled in Asia, which is logically consistent with
family history.
The N/R subgroup B result is not accordant with the original hypothesis M subgroup
Q, which is reasonable because both 2 haplogroups have high possibility to discover
in Asia, especially East Asia [5][12].
A relative research paper Mitochondrial haplogroup B is negatively associated with
elite Korean endurance athlete status focuses on the mutation genes commonly
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among subgroup B in Korea. It mentions that mitochondrial DNA sequence in
subgroup B probably causes oxidative phosphorylation deficiency during aerobic ATP
energy derivation. The experiment is held with 132 elite Korean athletes separated
into sprint/power group and endurance/middle-power group and 246 controls. Their
DNA is analyzed, and 246 controls have higher frequency of mutation from T to C at
position 16189. The conclusion is driven: transition in subgroup B has negative
effects on elite Korean endurance status [13][14].
With the confirmation of haplogroup classification, the mkj5122 nucleotide sequences
are run in Blast Search to identify the DNA. According to figure 4 and 5, the percent
identity for the first sequence is 91%. The blasted query have 3 mutations from G or A
to C differing from the subject sequence.

Figure 5: DNA Blast Graphic Summary

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Figure 6: Query and Subject Comparison
The information of mostly matched sequence rCRS is published in Reanalysis and
revision of the Cambridge reference sequence for human mitochondrial DNA. This
article describes the origin of CRS and mentions the polymorphism of DNA. Some
ambiguous nucleotides are edited and replaced to build the revised CRS (rCRS) used
widely nowadays [15].
The experimental errors like gel electrophoresis non-sense DNA travel distances can
be eliminated or avoided by examination lab facilities and repeated steps. Some
contaminant might appear in the DNA columns and PCR tubes, thus the equipments
should be cleaned and sterilized carefully. At the aspect of sequencing comparison,
experimenter’s DNA sequence is not long enough to analyze. There are only 2
haplogroup markers which might provide incomplete or incorrect information on
haplogroup classification; the more detailed and accurate distinguish can be
proceeded in further study.
Reviewing the primary goal for this experiment, the conclusion is made: the
experiment’s DNA sequence (mkj5122) is classified into N/R subgroup B, which is
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considered, originated in Africa, migrated to Europe, and eventually settled in East
Asia.

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References
[1] Finnilä, Saara, Mervi S. Lehtonen, and Kari Majamaa. "Phylogenetic Network for
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[2] Mckinney, Emily A., and Marcos T. Oliveira. "Replicating Animal Mitochondrial
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[3] BIOL 220W: Populations and Communities Laboratory Manual-Spring 2016.
Penn State University, 1 Mar. 2016
[4] Bandelt, Hans-Jürgen, Anita Kloss-Brandstätter, Martin B. Richards, Yong-Gang
Yao, and Ian Logan. "The Case for the Continuing Use of the Revised
Cambridge Reference Sequence (rCRS) and the Standardization of Notation
in Human Mitochondrial DNA Studies." 2013. Journal of Human Genetics J
Hum Genet 59(2): 66-77.
[5] "More about Haplogroups." 23andMe Customer Care. N.p., n.d. Web. 01 Mar.
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[10] Haplogroup Markers:
http://mitomap.org/bin/view.pl/MITOMAP/HaplogroupMarkers
[11] Simple Tree Mitomap (Web)
http://www.mitomap.org/pub/MITOMAP/MitomapFigures/simple-tree-mit
omap-2012.pdf
[12] Kong, Qing-Peng, Yong-Gang Yao, Chang Sun, Hans-Jürgen Bandelt,
Chun-Ling Zhu, and Ya-Ping Zhang. "Phylogeny of East Asian
Mitochondrial DNA Lineages Inferred from Complete Sequences." 2003.
The American Journal of Human Genetics. 73 (3): 671-76. Web.
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[13] Kim, Ki Cheol, Han Jun Jin, and Wook Kim. "Mitochondrial Haplogroup B Is
Negatively Associated with Elite Korean Endurance Athlete Status." Genes
Genom Genes & Genomics 34.5 (2012): 569-73.
[14] Kivisild T, Tolk HV, Parik J, Wang Y, Papiha SS, Bandelt HJ and Villems R
“The emerging limbs and twigs of the East Asian mtDNA tree” 2002. Mol.
Biol. Evol. 19: 1737-1751.
[15] Andrews,R.M., Kubacka,I., Chinnery,P.F., Lightowlers,R.N.,Turnbull,D.M. and
Howell,N. “Reanalysis and revision of the Cambridge reference sequence
for human mitochondrial DNA” 1999. Nat. Genet. 23 (2), 147

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