This lab report was written in April 2015 for a Genetics Laboratory assignment on mitochondrial DNA. Downloadable version of this report is available here.
Mitochondrial DNA (mtDNA) is DNA that is only found in mitochondria, and differs from the nuclear DNA in the normal genome. This lab examined mtDNA sample sequences from students in the class, and compared those sequences against the rest of the class, as well as the Chimp and the Neanderthal. BLAST was used to find top matches to the D-loop mtDNA sequence. Comparing sequences allows common nucleotides to be identified, and the more nucleotides that are alike within a sequence, the more genetically similar two organisms are. The average divergences and substitutions were calculated, allowing a rate of substitution to be calculated. With these rates and the commonly accepted divergences (5,000,000 years between Chimps and Humans; 200,000 years between Neanderthals and Humans), divergence times were calculated. Of course, the two Humans should have the smallest divergence time, with an average sequence divergence of 0.017, and a divergence time of 504,002.37 years. The results of these calculations showed that the Human and the Neanderthal diverged more recently than the Human and the Chimp, and are therefore the most alike. This qualifies the Neanderthal to be the most recent common ancestor (MRCA) of the modern human, upholding the displacement model.
Most early scientific education focuses on nuclear DNA, but there is also another type of DNA: mitochondrial DNA (mtDNA). Both are double-stranded and encode for genes, but mtDNA has no introns, codes for less genes, has different codon sequences, has a much higher mutation rate because there is no proofreading, is only inherited from the mother, and has only one or two bases between genes (Mitochondrial Disorders, 2015). This mitochondrial DNA is circular, and the genes within it are much closer to each other, with few introns. There is still a noncoding region on both sides of the “0” position in the genome; this region is called the D-loop. This name is a reference to the loop (or bubble) that forms during DNA replication. The D-loop is the main regulatory region of the circular DNA, and we look at it because its high rates of mutation make it easy to identify similar sequences (Pesole, Spisa, Preparata, Saccone, 1991). The mitochondrial DNA mutates at a much higher rate than nuclear DNA due to its increased exposure to free radicals, and that makes it easier to determine how similar species are, according to how many mutations they share.
MtDNA is only passed maternally. Though males have mtDNA, when it comes time to reproduce, their mtDNA either doesn’t make it from the sperm into the egg, only negligible numbers enter, or they enter and are marked for destruction (Groleau, 2002). This means that mtDNA is a good way to test maternity, but paternity testing is impossible with mtDNA. Because mtDNA is so useful in tracing ancestry, databases such as BLAST have been created as a library of many mtDNA sequences. Such databases allow scientists to look for homologies between different species and/or organisms by comparing the two sequences against each other and looking to see how many of the bases are the same. Sequences that match very closely are more recently related to each other than sequences with few lower correlation values, more commonly known as percent identity.
As earlier mentioned, every time species diverge from a common ancestor, they acquire a new mutation. These mutations continue to occur, and a molecular clock can be established by looking at this relationship between time and how many mutations are in the mtDNA. This clock is calibrated off the fact that humans and chimps diverged about 5,000,000 years ago. The clock then allows divergence times to be calculated, given the substitution rate and how many bases are homologous (Molecular Clocks, 2006). MtDNA has also been used to trace ethnographic and geographic origins, and known sequences of a certain geographic origin are now in databases such as the NCBI database, allowing mtDNA sequences to be compared against them, thus identifying where the organism sampled originated.
As origination goes, there are two main theories on how modern humans evolved, and where they began. The multiregional is the first model, and it states that people developed and evolved independent of each other across the world. The multiregional puts the most recent common ancestor (MRCA) 1.5-3 million years ago. The displacement model is the second model, and is also known as the “Out of Africa” model. This theory believes that there was one original population in Africa that populations migrated away from and the groups thus all developed at the same time. This theory suggests that humans diverged 200,000-500,000 years ago, which would support the idea that humans and humans diverged more recently than humans and Neanderthals. Using the divergence times from the molecular clock makes it possible to determine whether the class data supports the multiregional or displacement model.
Materials and Methods
To extract mtDNA, swish 10mL of a 0.9% NaCl saline solution for 30 seconds and spit into a cup. Swirl and add 1.5mL (3mL total) to two labeled 1.5mL tubes. Microcentrifuge at maximum speed for 5 minutes. Remove, and remove the supernatant. Resuspend the pellet in the saline solution, and add to a labeled 0.5mL tube containing 100uL of the 10% Chelex solution. Vortex. Heat sample for 10 minutes using thermocycler set at 100°C. Microcentrifuge for 1 minute. Transfer 50uL to a new, labeled 1.5mL tube and store for PCR
To perform PCR, add 22.5uL of primer/ddH2O mix to a 500uL PCR tube with a Ready-to-Go PCR Bead and tap to dissolve. Transfer 2.5uL of the genomic DNA to the tube and tap to mix. Store on ice, and then run the thermal cycler for 30 cycles. Each cycle includes initial denaturing at 94°C for 2 minutes, denaturing at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extending at 72°C for 6 minutes. After the 30 cycles, the final extension will be at 72°C for 6 minutes, followed by an indefinite hold at 4°C.
Perform gel electrophoresis and DNA sequencing (performed by instructors). This will determine is the DNA is present.
To find sequences similar to the mtDNA sequences found, use NCBI BLAST, specifically nucleotide blast. Use the settings nucleotide collection (nr/nt), Homo sapiens, and somewhat similar sequences (blastn). Paste DNA sequence into query sequence box, and press the BLAST button. Identify the two sequences with the greatest similarity (highest “Max score” value), record accession numbers and the nucleotide positions associated with the similar regions. Record the number and percent of bases that were identical.
Using sequence comparison data, calculate the average proportional divergence (pa) and the number of substitutions per site (Kn = -ln[1-pa]) for human vs. Neanderthal, human vs. Chimp, and human vs. human. Then, obtain the rate of nucleotide substitution (substitutions per site per year) and use that rate to calculate the divergence times for those comparisons, using the fact that substitutions per site divided by substitutions per site per year gives years.
Using BLAST, the top two matches were found for the D-loop sequence 3-6. The Accession Number, E-score, percent identity, and length of match for each match can be found in Table 1. Match 2 was from the haplogroup M3f.
|Table 1: Initial Matches to D-loop DNA Sequence|
|Accession #||E-score||% Identity||Match Length|
|Match 1||KJ446515.1||2e-174||99%||345 bases|
|Match 2||JQ045085.1||2e-174||99%||345 bases|
The class sequences were compared to Neanderthal and Chimp sequences, as well as to each other. The average proportional divergence (pa) and the average number of substitutions per site (KHX) were found for the three comparisons. The results of these comparisons can be seen in Table 2.
|Table 2: Average Proportional Divergences and Substitutions per Site|
|Avg Proportional Divergence||Avg # substitutions/site|
|Human v. Neanderthal||0.047||0.048|
|Human v. Chimp||0.155||0.169|
|Human v. Human||0.017||0.017|
The human-chimp divergence time is 5,000,000 years; dividing the average number of substitutions per site for the human-chimp by this time gives substitutions per site per year. Using that rate of substitution, divergence time from the most recent ancestor to the human can be calculated. To do this, the average number of substitutions per site must be divided by the rate of nucleotide substitution, giving an answer in terms of years. The same process was carried out for the Neanderthals and modern human, with an established divergence of 200,000 years. These results are recorded in Table 3, as is the time since the two classmates shared a common ancestor.
|Table 3: Divergence Times|
|Substitutions Per Site Per Year||Divergence Time|
|Human v. Neanderthal||8.558E-08||560,878.71 years|
|Human v. Chimp||3.373E-08||–|
|Human v. Human||–||504,002.37 years|
A sample calculation of the values in Table 3 can be done for the Human v. Neanderthal:
Substitutions Per Site Per Year= Substitutions Per Site (from Table 2)/Established Date of Divergence
=8.558E-08 Substitutions Per Site Per Year
Divergence Time= Substitutions Per Site/Substitutions Per Site Per Year
=560,878.71 Years Since last common ancestor
A low value for average substitutions indicated that the species have not diverged very much, as species diverge and evolve due to increased substitutional mutations that cause changes in phenotype. As shown in Table 2, the human and human match had the lowest average number of substitutions per site because they are the same species. The human and Neanderthal have a greater substitution value, and the human and Chimp have the highest. This indicated that the human and the Chimp mtDNA sequences differ the most of all three comparisons, making them the least similar. The fact that they are least similar corresponds to the fact that they have an older divergence than the Neanderthal and the human. As seen in Table 3, the human and the Neanderthals’ last common ancestor was roughly 560,000 years ago. This indicates that the human and the Neanderthal are more closely related than the human and the Chimp, which diverged 5,000,000 years ago. This indicates that the displacement model of human evolution is more consistent with the data because it is a more recent divergence. Though it is more true to the displacement model, the calculated estimate of the divergence time is double the accepted 200,000 years. One explanation for this difference is that this lab only looked at mtDNA, not chromosomal DNA mutations. Calculating these divergence values reinforced my previously held understanding of evolution, as depicted in timelines of the evolution of the Homo genus (The Genus Homo, 2011).
Groleau, Rick. 2002. Tracing Ancestry with mtNDA. Nova Online. http://www.pbs.org/wgbh/nova/neanderthals/mtdna.html (Accessed April 9th, 2015).
Mitochondrial Disorders. 2015. Washington University in St. Louis. http://neuromuscular.wustl.edu/mitosyn.html#general (Accessed April 8, 2015).
Molecular Clocks. 2006. The University of California Museum of Paleontology, Berkeley, and the Regents of the University of California. http://evolution.berkeley.edu/evosite/evo101/IIE1cMolecularclocks.shtml (Accessed April 8, 2015).
Pesole, G., E. Sbisa, G. Preparata, and C. Saccone. 1991. The Evolution of the Mitochondrial D-Loop Region and the Origin of Modern Man. Molecular Biology and Evolution. http://mbe.oxfordjournals.org/content/9/4/587.full.pdf (Accessed April 8, 2015).
The Genus Homo. 2011. Archaeologyinfo. http://archaeologyinfo.com/genus-homo/ (Accessed April 7, 2015)