Miranda and Chiemerie

Three bio powerhouses

Why the powerhouse of the cell?

As you may know, the mitochondria is infamous for its title as powerhouse of the cell, and while we certainly appreciate it for its role in energy conversion, we were intrigued by the evolutionary background and potential for ancestral history we saw in mitochondrial DNA -- after all, we were curious about our own haplogroups!

goal video.mov

Some power-ful results

An auditory learner? Just want a quick summary?

Hear us chat about our goals, process, and results in this video!

DYO adv bio methods vid.mp4

Does this gel with you?

Learn how to do gel electrophoresis with our methods video :)

What's mtDNA? A lil Background + Methods

mtDNA (or Mitochondrial DNA) is a piece of DNA that is passed down matrilineally (meaning from one’s mother to their offspring. mtDNA--as the name suggests--is stored within the mitochondria, and exists outside of the nuclear genome. mtDNA is thought to have evolved completely separate from nuclear DNA, and to have originated from bacteria that early eukaryotic organisms engulfed. The DNA itself only has 16, 569 base pairs and encodes for a mere 13 proteins, all of which are fittingly involved in oxidative and phosphorylation processes that happen within the mitochondrion. Although mtDNA may seem small and relatively negligible, mtDNA possesses unique qualities that makes it a big area of attention in the field of human evolutionary biology.

Because of the way mitochondrial DNA persists across generations, it provides a useful tool for examining human evolution as well as the human diaspora out of East Africa. Mitochondrial DNA is also five times as prone to mutating than nucleic DNA, with the mutations usually being in the form of single nucleotide polymorphisms. Single nucleotide polymorphisms (SNPs) are a difference in a DNA sequence that represents only that of a single nucleotide. By comparing SNPs on different people’s mtDNA, scientists have been able to categorize those who share the same/similar mtDNA SNPs into singular haplogroups, those haplogroups consisting of people who share a common ancestor. In our experiment, we sequenced various individuals’ hypervariable regions on their mitochondrial DNA and sequenced them in order to gain a better understanding of each individual’s haplogroup and genetic lineage, as well as to examine any potential haplotypic similarities among our classmates.

To do so, we first amplified each of the 12 tested individuals’ Hypervariable regions on their mitochondrial DNA by means of Polymerase Chain Reaction (PCR). Afterwards, did gel electrophoresis on a bit of each person’s amplified DNA to confirm our PCR had amplified at all/amplified the correct section of DNA. Once we confirmed that each individual’s HVR had been correctly amplified, we sent the samples off to a lab to be sanger sequenced. Once our results came back, we used the sequence query on mitomap to approximate each individual’s mitochondrial haplotype. We also calculated the percent difference between each individual’s sequence with another individual, providing us with two different metrics to compare haplotypic similarity amongst the class

The results of our lab not only mainly reaffirmed the ethinic/racial identities of members of our classroom , but also provided us with interesting information regarding ancestral migration, potential common ancestry, SNP delineation and haplotypic rarities among specific members of our class. In many ways, studying our classroom’s current mitochondrial DNA sent us searching into the past to learn more about the beginnings human civilization as we now know it!

Graphical Abstract of Our Experiment

Snapshots of our process:

Comparing The Data pt. I

We used Benchling to study the results of the Sanger sequencing and to measure the precision with which each set of DNA was really reported. We looked for Quality Indexes of above 20, which led to the cropping of two particular sets -- 1 and 12 -- and some hesitancy surrounding sets 7 and 8 which had relatively low quality indexes. In Benchling we were able to compare specific SNPs and to count the number of differences between any two participants.

Comparing The Data pt. II

We took the differences that we counted in Benchling and then mapped the percent difference between each of the participants. We note that the percents are all relatively low except in the cases of 7 and 8 which are a little higher, likely due to the lower quality indexes and thus greater potential for the misclassification of nucleotides. We also note the 0% difference between Miranda and her mom Kate, which makes sense given that mtDNA is passed maternally.

An Early Signal of Success

The bands apparent in each lane of this gel electrophoresis signal a successful PCR reaction as we can see the DNA was successfully replicated at the particular length. This success gives us the greenlight to send off our DNA samples for sequencing.

Determining Our Haplogroups

We used a website called MitoMap to help us predict the haplogroups of each participant by inputting their sequenced nucleotides. In this example, the participant is predicted to have a L1b mitochondrial haplogroup, as seen in the table at the bottom of the picture.

Where in the World Do We Go From Here?

Mitochondrial haplogroups can be used to trace ancestry across the globe with different groups corresponding to different regions. Additionally, each haplogroup comes with it's own 'expansion time' which explains when that branch formed.

Results Discussion + Evolutionary Tie Backs

Our results gave way to many cool discoveries, one being that not all Haplogroups associated with indigenous Africans date back to before the migration out of Africa , despite East Africa being the point from which Homo Sapiens left the continent to populate the Earth (most models predicting the first migration dates 270,000 years ago). This was evident in Chiemerie and person 6’s haplotype: L-1B. This haplotype dates back by only 10,000 years, a number much smaller than the it’s sister haplotype L-1C time, being 85,000 years (both L1B and L1C originated in central Africa).

We also had the chance to sequence Miranda and her mother’s DNA, and for the parts that were not poorly sequenced (we had to shorten both sequences to around 200 bp for each due to faulty sequencing towards the end) were exactly the same! This simply supports the idea that mtDNA is passed matrilineally; however, since we could not look at the full HVS sequence on either of them, our data cannot confirm or deny that there is no mtSNP discordance (though highly rare despite how prone to mutations mtDNA is) between Miranda and her mom. The lack of full sequences from both Miranda and her mom also made it hard to pin down their correct haplogroups; while both of theirs came out as H-2A, we still cannot be as sure about their haplogroup categorization.

Another interesting piece of our data was being able to trace specific early human migratory behavior out of Africa. For example a student of South Asian descent whose mtDNA was sequenced was given haplogroup L3B, a haplogroup that dates back 70,000 years, when early members of this clade made the most recent out of Africa migration called the Southern Disposal. The group of humans that left this route left from the horn of Africa, entered west Asia, to migrate along the coast of South Asia, supported by the student’s ethnic identity. Another interesting fact about L3 is that, unlike L-1-6, L3 is the progenitor to all the other non African Haplogroups. At the same time L3A (an earlier subclade than l3B) is ethnically associated with people of East African descent, further supporting the Southern Disposal Theory Out of East Africa.

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