Natalie Cappelli

March 22, 2016

TA: Chelsea Hudson

Section: 220M

Human Genetic Variation: Tracing Ancestral Maternal Lineages

Introduction:

A human cell is composed of two genomes; one of which is the nuclear genome that

consists of linear chromosomes in the nucleus, and the other being the mitochondrial genome

that is found in the mitochondria present in every cell. There are hundreds and sometimes

thousands of mitochondria present in every human cell, each one containing several copies of its

genetic code, also known as mitochondrial DNA. Therefore, the rate of evolution for

mitochondrial DNA is greater than the nuclear genome due to small changes, or polymorphisms,

created by the coping process (FTDNA Learning Center Maternal Lineage Tests). When taking

a closer look at human mitochondrial DNA, the control region is the most adaptable region,

specifically the hypervariable regions, HV1, because it is the most random part of the genome

(Burpee et al., 2017). Additionally, the hypervariable sites in human mitochondrial DNA are

readily identified when studying evolution and generally represent mutational hotspots

(Stoneking, 2000). In fact, the high number of nucleotide polymorphisms or sequence variants in

the hypervariable regions can allot for discrimination among individuals and other biological

samples (Johnson). As a result, mitochondrial DNA samples can be taken from extremely small

samples and then amplified using the Polymerase Chain Reaction (PCR), targeting the HV1

section of the DNA. Moving forward, human mitochondria are primarily inherited from an

individuals mother (Burpee et al., 2017). Thus, human geographic migration throughout time
can be studied through the analysis of the mitochondrial genome that follows the maternal

lineage.

Tracing ones mitochondrial genome through time and discovering the lineage in which it

originated is important in determining how the human population is connected as a species.

Mitochondrial DNA can be used to represent how travel and movement from generation to

generation affects genetic diversity in the long-run. Specifically, the polymorphisms found in a

region of HV1 can be used to find the particular haplogroups from which it originated, and then

can be traced back to the origin of ones mitochondrial DNA. The focus of this study was to

analyze our own personal mitochondrial DNA sequences based on gel electrophoresis and PCR

in an effort to trace back our maternal lineage to its origin. As a person with a primary Italian

background, I hypothesized that my mitochondrial DNA originated in Europe.

Materials and Methods:

The material and methods were followed directly from A Laboratory Manual for Biology

220W: Populations and Communities (Burpee et al., 2017).

DNA Extraction

To prepare this lab, the inside of my cheek was swabbed using a cotton swab to collect

cells. The DNA was extracted using the procedure presented in A Laboratory Manual for

Biology 220W: Populations and Communities (Burpee et al., 2017). The cells were incubated in a

hot block until they were removed at 11:00 am, on the 26th of January 2017.

PCR

The DNA was then extracted and isolated after multiple spins in the centrifuge before

performing a PCR amplification of the HV1 mitochondrial sequences using specific primers.

PCR was performed after extracting and isolating the DNA in my sample following the
procedure found in A Laboratory Manual for Biology 220W: Populations and Communities

(Burpee et al., 2017). My DNA concentration came out to be 105 ng/l, so therefore 49 l of

PCR master mix was added along with 1 l of sample DNA.

Gel Electrophoresis

Preparations were then made to run the gel electrophoresis following the procedure

located in A Laboratory Manual for Biology 220W: Populations and Communities (Burpee et al.,

2017). Once the agarose hardened, the wells were filled with a DNA ladder, two wells of my

sample, and two wells of my partners sample, and a negative control, in that order respectively.

PCR Preparation for Sequencing

Moving forward, the PCR products were primed in order to prepare the DNA for

sequencing following the procedures listed in A Laboratory Manual for Biology 220W:

Populations and Communities (Burpee et al., 2017). The results from our DNA sequencing were

downloaded to Canvas in order to use Mega to visualize and analyze our DNA sequence data to

determine our maternal lineages.

Analyzing Sequences

After examining our DNA sequence, MITOMAP was used to find the reference sequence

position and also the haplogroup designations for each site. A BLAST search was then conducted

in order to locate the differences between our queries and the reference sequence, rCRS.

Results:

My DNA concentration came out to be 105 ng/l, so therefore 49 l of PCR master mix

was added along with 1 l of sample DNA.

The results from my gel electrophoresis came out to be very transparent when placed

under the UV light. Although it was clear that there was DNA present in my samples, it was a
very small amount, and as a result my trace sequence came out to be messy at the beginning.

Therefore, my trace sequence had an array of overlapping peaks in which the base pair could not

be determined. However, after site 77, the base pairs were able to be sequenced because the

peaks became very clear. When investigating my sequence, it was clear that my DNA sequence

did not match with the rCRS sequence until site 72.

Figure 1. Picture of my gel electrophoresis.

My sequence showed that two known polymorphisms had occurred. The first was seen at

site 192, which was equal to 16166 as a reference sequence position. At this site, the A base pair

found in the rCRS sequence was deleted in my sequence. The second mutation was found at site

369, which corresponded to reference position 16343. At this site, the A base pair found in the

rCRS sequence was substituted to a G base pair in my sequence. Both mutations found in my

HV1 region were known mutations, but came from no known haplogroup sites. As a result, it

was summarized that the designated sites could fall under any of the following, L, M, N, P, H,

UK-group, or S.
 Alignment Reference Mutation rCRS Is this a known Haplogroup
Position Sequence to your polymorphism Designation(s)
Position sequence for this site
(tentative
indicated
haplogroup in
bold)
192 16166 A to - Yes L, M, N, P, H,

UK-group, S
369 16343 A to G Yes L, M, N, P, H,

UK-group, S
Figure 2. Mutation Information

The results from my BLAST search showed that 99% of my sequence in the list of

sequences of returned. There were approximately 4 differences between query and the closest

match, making 372 out 376 correct. As Figure 3 shows, two mutations appeared in my search,

one being a deletion and another being a substitution. The other two errors occurred as a result of

the two undetermined base pairs near the beginning of my DNA sequence (Andrews et al., 1999).

Figure 3. Results of my BLAST search (Andrews et al., 1999).

 Figure 4. Human mtDNA migrations based on haplogroup sites
(MITOMAP).

Discussion:

As stated in results, when I entered my reference sequence positions of my two known

polymorphisms into a MITOPMAP search, I found that there were no known sites. However,

although there were no known sites, a list of default haplogroup designations were established

including L, M, N, P, H, UK-group, and S (HaplogroupMarkers). I found that my maternal

lineage originated in Europe, specifically from site N, although there was equal support from all

haplogroup designations found in my MITOMAP search (Figure 4).

Despite the fact that both of the polymorphisms found in my HV1 region accounted to no

known sites, the sites that defaulted to represent those in my case were used in a process of

elimination. Because I know that my mothers grandmother immigrated to America from Italy, I

used the haplogroup designation N to follow my lineage through time. The arrows on the map

showed that N traveled from Europe to America which applies to my situation due to the fact that

I know my great-grandparents immigrated to Greensburg, Pennsylvania. This is also because site

N was included in my options under the no known sites category, and I know that N on the

map had originated in an area that my ancestors had immigrated from. Therefore, site N

confirmed that my maternal lineage originated in Europe, correlating with my original

hypothesis.
 Although the findings paralleled where my mothers family came from, possible sources

of error could include cross contamination of the DNA or losing some of my DNA during the

process which could be why my trace sequence originally came out messy so that few base pairs

could be identified until later site. After completing the study and determining the origin of my

maternal lineage, further studies can be conducted in order to further understand where my

ancestors came from. Therefore, this could be done by tracing my fathers maternal lineage. In

order to do so, my fathers DNA would have to be used to trace his mothers lineage in

comparison to using my DNA to trace my maternal lineage due to the fact that mitochondrial

DNA is inherited through the mothers line only (FTDNA Learning Center Maternal Lineage

Tests).

References:

Allele Search. (n.d.). Retrieved March 15, 2017, from

http://mitomap.org/bin/view.pl/Main/SearchAllele.

Andrews, R.M., Kubacka, I., Chinnery, P.F., Lightowlers, R.N., Turnbull, D.M., and N. Howell.
 Reanalysis and revision of the Cambridge reference sequence for human mitochondrial

DNA. 1999. Nat. Genet. 23: 147.

Burpee, D., Hass, C., Ikis, D. and K. Richter, eds. A Laboratory Manual for Biology 220W:

Populations and Communities. 2017. Department of Biology, The Pennsylvania State

University, University Park, PA.

FTDNA Learning Center Maternal Lineage Tests. (n.d.). Retrieved March 10, 2017, from

https://www.familytreedna.com/learn/dna-basics/mtdna/

HaplogroupMarkers < MITOMAP < Foswiki. (n.d.). Retrieved March 15, 2017, from

http://www.mitomap.org/bin/view.pl/MITOMAP/HaplogroupMarkers.

Johnson, S. (n.d.). mtDNA Basics. Mitotyping Technologies. Retrieved Feb. 28, 2017, from

www.mitotyping.com.

MITOMAP < Foswiki. (n.d.). Retrieved March 15, 2017, from

http://www.mitomap.org/MITOMAP.

Stoneking, M. (2000). Hypervariable Sites in the mtDNA Control Region Are Mutational

Hotspots. American Journal of Human Genetics, 67(4), 10291032

Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics

Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599.