Spring
A view at the genetic variation in human populations
Dominic Galanti
BIOL 220M
Pennsylvania State University
TA: Andrew Fister
Due: March 24, 2014
Human Genetic Variation
14
Human Genetic Variation
Galanti,1
Introduction
Human origins have been traced to central Africa. As the species dispersed
throughout the globe mutations developed in different groups of individuals,
characterizing the humans from each particular region. These mutations result in a
phenotypic variation in the population. This experiment investigates the human
control regions of the mitochondrial DNA. Mitochondrial DNA is used as there are
many mitochondrial organelles in each cell allowing for more DNA, thus resulting in
more successful experiments. Also this DNA is commonly used in haplogroup
determination in prior experiments. The hypervariable regions, HV1 and HV2, show
a greater difference from individual to individual. Through investigation of HV1 a
comparison between data sets could be performed to locate origins of the human
from each data sample. By gathering data of genetic variation in humans to compare
the scientific community will have a better understanding of what traits individuals
from different backgrounds may have including appearance and disease risk. The
region being examined, HV1, is known to have more substitutions in the nucleotide
sequence when compared to other regions in the sequence.1 If DNA samples are
collected and sequenced from individuals then the results will show a number of
mutations differing with each individual, which provides data to each individual of
their genetic ancestry.
Materials and Methods
First DNA was obtained from two individuals using a cheek swab procedure
to ensure proper DNA quantity. Once on the cheek swab the cheek skin cells were
Human Genetic Variation
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transferred to a microcentrifuge tube where buffer ATL and proteinase K were
added. Tubes were placed on a hot block at 56° C to lyse the cells and expose the
DNA.
After one week the DNA was extracted from the tube and washed to remove
impurities. A DNeasy Spin Column was given buffer and ethanol and the DNA
sample was transferred to the spin column. The spin column was centrifuged at
8000 rpm for one minute. This process was repeated keeping the spin column and
replacing the collection tube for two additional washes one with buffer AW1 and
one with buffer AW2. Lastly the spin column was washed with Buffer AE and a
microcentrifuge tube with the collected DNA was produced. A NanoVue
Spectrophotometer was used to provide data on the quantity of DNA in the tube.
Five PCR tubes were prepared for PCR, two with DNA from each individual
and one negative control with sterile water. Equal amounts of DNA sample and
sterile water were added to each of the tubes. These tubes were loaded into a PCR
machine and once the PCR was run they were stored frozen for one week.
After an additional week the PCR products were allowed to warm up to room
temperature and then run through gel electrophoresis. In addition to the five tubes
of PCR product prepared with loading dye, DNA ladder was added to the gel. Gel
electrophoresis was run and the gel was photographed under UV light.
Buffer PB was added to the PCR products and transferred to a spin column
where it was centrifuged and then washed again with buffer PE and centrifuged. The
PCR products were collected by centrifuging sterile water through the spin column
and collecting the flow through. Lastly the flow though was loaded and sent to be
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sequenced. Instead of sample from only one of each DNA sample, tubes of like DNA
were combined in order to have a large enough volume of sample and sent to be
sequenced one with the label dag5441 and the other vaco528
Results and Discussion
Figure one shows the results of the PCR products and negative control run
through gel electrophoresis. This was a failed gel procedure as the only visible
bands are of the ladder. The ladder provides a comparison to the PCR products but
since there are none to be shown it is irrelevant data. The lack of results from the
other wells could be a lack of DNA concentration in the solution as one DNA
concentration was 20 ng/l and the other was 58.5 ng/l under the NanoVue
Spectrophotometer or the DNA may not have mixed with the loading dye well
enough.
Figure two illustrates the mutations found in the DNA of the dag5441 sample.
The two known mutations lead to the understanding that the lineage of this
individual came from and area in western Asia. With only two known mutations the
haplogroup determination is less valid but because the N/R group is found in both
mutations that is an accurate estimation of an early lineage followed by the
branching off into subgroup F because one of the mutations is only found in this
subgroup. This haplogroup determination makes relatively good sense as my
ancestry is from the area of Italy to Czechoslovakia and up towards Poland. This
location is the east of Europe and very close to western Asia where these
haplogroups are found. Three additional potential mutations are not applicable
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because the sequencer could not determine what base was being expressed at that
site. Therefore this section of data could not be compared to the reference sequence.
Towards the end of the sample sequence there was and insertion causing the
following bases to be one base out of order in compared with the reference
sequence. None of these mutations towards the end were known polymorphisms so
it can be understood that this is an insertion and not a point mutation.
A blast search of the dag5441 sample shows a 98% identity to a related
sequence.3 The differences between these two sequences were caused by three
undetermined bases and one insertion of an adenine base into the dag5441
sequence.
In conclusion this data shows that there is differentiation in the human
population due to mutations in the mitochondrial DNA. These mutations are most
commonly the result of a substitution but other exceptions such as insertions exist.
The haplogroup of my DNA sample is the N/R group with a probable subgroup of F.
By the blast search it is shown that other sequenced samples exhibit different
mutations, thus showing that the human population has different genetic makeups
depending on one’s lineage.
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Figure 1: Gel Electrophoresis under UV light
Ladder
Dag5441
Dag5441
Vaco528
Vaco528
Figure 2: Mutations in Sample dag5441 from Reference rCRS
Alignment
Reference
Mutation from Known
Haplogroup(s)
Position
Sequence
reference to
Polymorphism?
Position
sample DNA
330
16304
TC
Yes
N/R Subgroup
F
337
16311
TC
Yes
M Subgroup Q,
N/R Subgroup
HV-group and
UK
359
16333
N
N/A
N/A
360
16334
N
N/A
N/A
361
16335
N
N/A
N/A
444
16418
TA
No
N/A
445
16419
CT
No
N/A
446
16420
AC
No
N/A
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References
Wakeley, John. 1993. Substitution Rate Variation Among Sites in Hypervariable
Region 1 of Human Mitochondrial DNA. Department of Integrative Biology,
University of California, Berkely, CA 94720. Journal of Molecular Evolution 37:613623.
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.
Kogelnik, A.M., Lott M.t. Submitted 8/24/06 Mitomap.org, Center for Molecular and
Mitochondrial Medicine and Genetics. University of California, Irvine. CA 926973940