Extraction, Amplification and Electrophoresis of Human Mitochondrial DNA

   Imagine that we could trace our family tree back to the first human ever to set foot on earth. Well actually, it doesn't take much of an imagination, as the study of mitochondrial DNA may offer us just that possibility. From an early age, everyone learns what DNA is. Mom and dad pass some stuff to their children that makes them look and act alike. But a lesser known fact is that there is a different type of DNA that we obtain solely from our mothers.
   In all eukaryotic organisms are organelles called mitochondria. It is theorized that mitochondria were once their own organism but were engulfed by a more sophisticated eukaryotic cell. Mitochondria utilize oxygen from the atmosphere to create energy that fuels the functioning of the cells, making it one of the most vital organelles in our bodies.
   Mitochondria actually carries its own set of DNA that codes for the machinery that allows it to synthesize energy (ATP). This DNA comes directly from our mothers, creating a lineage based on nearly-identical mitochondrial DNA, differing only due to mutation. But why do we procure this DNA just from our mothers? Sperm, hosting the male genome, must be small and elusive to make its way to the female egg, so it carries little more than the necessary DNA, with just enough mitochondria to power its journey. The egg, on the other hand, contains all the typical organelles. Therefore, the mother is the one to contribute its cytoplasm and organelles, including the mitochondria, to the zygote.

   The mitochondrial (mt) genome was sequenced far before the human genome because it only contains 16,569 nucleotides and 37 genes. The mt genome has few introns, but it does have one long non-coding stretch that is highly mutative. The region's supervariable quality creates SNPs that help establish familial relationships. Based on rates of mutation, scientists determined that the "mitochodrial Eve" first appeared 200,000 years ago in Africa- the origin of modern humans. The non-coding control region is especially useful because each cell contains hundreds of thousands of copies of each mt gene.
   For our experiment, we will use a template DNA instead of our own. First, we will add Chelex beads to the sample to break open the membrane and destroy ions that block PCR, and then we will separate the cells by vortexing. Next, we will amplify the extracted DNA through the process of PCR that I have described thousands of times. As a refresher, PCR requires DNA polymerase, primers, DNA nucleotides, and DNA ligase. It is essentially the rapid, controlled process of DNA replication. We will use specific primers that will target a segment of the non-coding region of mt DNA.
   Finally, we will run our mt DNA samples through gel electrophoresis, in which the DNA molecules will travel a certain distance, depending on their length (number of nucleotides).

MOMS

           GGel Electrophoresis

Our lab was a success, with everyone's mitochondrial DNA showing up on the gel. All our mt DNA strands were the same size, so they traveled the same distance on the gel. Because of this, we cannot make any differentiations between our mt DNA. For a small fee, however, we could send it off to be sequenced by a lab. We just might do that...

Teenage Mutant Electrophoresis

   DNA testing has opened a completely new realm of scientific possibilities. The process has evolved over the years from a tedious task in which a lone scientist charted nucleotides by hand to a routine procedure often rapidly performed by robots. The technique has numerous applications, including testing for evolutionary relationships, crime scene investigation, testing familial relationships, and individual testing for genetic predispositions for certain diseases.
   In our lab, we will be testing for a hypothetical disease, as disease testing is unethical in a high school environment. The gene for the "disease" we will be testing for comes in two separate forms: a long, sterile segment and a short, disease-causing segment. The short form is a recessive disorder, so we will need two copies (homozygous recessive) to have the predisposition. With Polymerase Chain Reaction (PCR) we will be able to target and amplify this specific gene, although in reality it is an intron that does not code for protein.
   There are several techniques for DNA testing, but we will be using gel electrophoresis. First, we need to extract our DNA, which we will take from cheek cells. After rinsing with a saline solution and collecting the cells, we will put the cells in a 95-degree Celsius water bath to break through the cellular and nuclear membrane. Hiding in the cytoplasm, however, are enzymes called DNAse that kill any DNA they find. This is a protection against foreign DNA, like from a virus, but it cannot tell the difference between viral DNA and its own genetic material. Therefore, we will add Instagene Matrix Beads that dismantle DNAse and allow us to extract our DNA, unharmed.
   This will be a tiny amount of DNA that won't be visible on a gel, so we must amplify the genetic material using PCR. PCR requires primer for our specific sequence, ample DNA nucleotides, DNA polymerase, and DNA ligase. During PCR, our DNA samples will rapidly replicate by a power of 2, giving us more than enough material to be seen in gel electrophoresis.

Lane 1 represents an individual with the genetic disorder (homozygous recessive). The recessive gene is smaller, so it travels farther in the gel. Lane 1, therefore, must have two copies of the recessive gene because there is only one band.
Lane 2 is homozygous dominant and does not have the disease. There is one band that did not travel very far, so it must represent two copies of the larger, dominant gene.
Lane 3 is heterozygous and doesn't have the disease. There are two bands, so it must have had one copy of each type of the gene.

 I'm not diseased!!! At first I believed I was diseased because I only saw one solid band designating the recessive gene, but there was a faint semblance of a band below it, meaning that I am heterozygous- a carrier. David and Taylor are diseased because they just have one far-traveling band. Schuyler is homozygous dominant, having zero copies of the disease gene.
There were some potential sources of error, but we avoided them. We loaded our gel properly, not puncturing the well or contaminating lanes with incorrect sample. Overall, this lab went perfectly.