Comparing Mitochondrion DNA

Introduction:

a) Mitochondria are the power plants of the cell. They provide energy, or ATP, for the cell through a process called oxidative phosphorylation. Without mitochondria, our cells would not be able to perform tasks necessary to live. Unlike the rest of the cell's organelles, mitochondria have their own unique set of DNA. One theory for why this is that mitochondria were once bacteria cells that became incorporated in a symbiotic relationship with eukaryotic cells. Mitochondria and bacteria are about the same size and both have similar, simple functions.



It is known that mitochondrial DNA can be traced back through the mother of each generation. A woman's egg contains all the organelles necessary for the embryo to survive, including all the mitochondria. A man's sperm has a lot of initial energy, but lacks mitochondria (thus, they eventually die). This way, when fertilization occurs and a baby is eventually born, all of the baby's mitochondria (and the mitochondrial DNA) were originally from the mother. This knowledge proves very useful for paternity testing and tracing family lines back for centuries.



b) The purpose of this lab is to extract our mitochondrial DNA and analyze it/compare it with other people at our lab table.
c) We will be using many similar techniques as the previous DNA extraction and "disease" testing lab. First, we will extract DNA from our cheek cells, using a saline mouthwash. Then we will concentrate the cells via centrifuge and add Chelex to the solution (Chelex binds to the ions released from the cells which inhibit PCR, thus allowing PCR to occur). The cheek cells will be lysed open via a hot water bath, releasing all the DNA. We will then add primers which specifically target a gene from the mitochondrial DNA. That way, when we run the DNA through PCR, only the mitochondrial gene will be amplified. Lastly, we will run the amplified DNA through gel electrophoresis so that we can get a visual on the DNA and compare our DNA results with the results of other people.
d) Since this lab is more of a comparison/knowledge activity than an experiment, there are no variables or controls. You could say the DNA is a variable, because obviously each person's DNA is different. However, we do not have a "control" gene to compare it to, so it makes no difference.

Testing for Genetic Diseases via DNA extraction, PCR, and gel electrophoresis

Introduction:

a) DNA testing is the extraction and analysis of the entire genome or, more commonly, a specific gene of interest. DNA testing has multiple applications. For example, it can be used for forensics, paternity testing, or, in this case, genetic disease diagnosis. Of course, we're not actually testing for a real genetic disease in class, but the principle is the same. DNA testing involves three main steps - DNA extraction, PCR, and gel electrophoresis - which will be discussed later in part c (techniques).
b) The purpose of this lab is to test ourselves for a "genetic disease". We will be utilizing all the techniques below in order to achieve this goal.
c) First, we start with DNA extraction - cheek cells will be swabbed and placed into a test tube containing a lysis buffer to help break open the cell/nuclear membranes. We will then put the cheek cells into a hot water bath of 95 degrees Celsius to further lyse open the membranes. Instagene matrix beads are used to kill DNAse, which would otherwise kill the exposed DNA. Second is PCR (polymerase chain reaction), which is used to make many copies of the DNA so that the DNA will be easier to test. The more copies, the more likely it will show up on the gel (part 3). A main ingredient of PCR is the primer, which targets the specific gene of interest. That way, we make copies of just the gene of interest (in this case, the "disease" gene), rather than the entire genome, which would be unnecessary. Third, we run the mass-produced genes on a gel. This process is called gel electrophoresis. The electric current run through the gel pulls the slightly negatively charged DNA toward the positive end - the smaller segments of DNA (in this case, the "diseased" gene is smaller than the "healthy" gene) will be able to work their way through the matrix faster and will thus travel farther toward the positive end. The larger pieces (the "healthy" gene) will be bogged down in the matrix and will not make it as far. Thus, gel electrophoresis has three potential outcomes:
1. Two bands that did not travel far --> indicates two "healthy" genes
2. Two bands that did travel far --> indicates two "diseased" genes
3. One band that did not and one band that did travel far --> indicates one "healthy" gene and one "diseased" gene.
Only outcome #2 will result in the expression of the disease, since the disease is recessive (both alleles must be recessive in order for the recessive genes to be expressed in the phenotype).

Results:

I do not have a picture of the gel results to post on this blab, but I can tell you that 3 of the people at my lab table tested positive for the "disease". In other words, each of us had 2 bands that travelled far and matched the bands of the "diseased" control gene. The fourth person's bands did not show up, so we cannot determine whether she was positive or negative. Luckily this "disease" is an intron, or else we'd all be in a bad spot!