Mitochondrial DNA

Background

In addition to nuclear DNA, each cell contains mitochondrial DNA, suitably named for its location in the mitochondria. Mitochondrial DNA is used to create and store ATP, which is used to power the cell. It is theorized that mitochondria may have been bacteria adapted into the cell in a symbiotic relationship; the bacteria would receive nutrients from the cell in return for energy created from oxygen.

^ Mitochondria_______________________

Mitochondrial DNA is inherited only from the mother, because while the sperm and oocyte both contribute chromosomes, only the oocyte contributes cytoplasm and organelles. Mitochondria also have a high mutation rate- approximately 10 times as often as nuclear DNA. These two features make it possible to trace ancestry by comparing the mutations present in individuals to those of their mothers, grandmothers, great-grandmothers, etc., etc. Work in the 1980s by Alan Wilson and others at UC Berkeley created a family tree, hypothesizing that all humans alive today evolved from a single ancestor. This "mitochondrial Eve" probably existed in Africa about 200,000 years ago.

^ a family tree_________________________

Mitochondrial DNA is extremely valuable in science. Because it is more prolific than nuclear DNA, it can be used in cases where much time has passed and DNA has been severely degraded.

Procedure

Skin cells are obtained either by a saline mouthwash or root ends from scalp, eyebrow, or arm hairs. A region is amplified using PCR, which cycles the DNA through three stages: first, a 94°C incubation to denature the DNA, a 58°C incubation to bond the primers to their complementary sequences, and a 72°C incubation for DNA polymerase to create complementary strands. Because of the large quantity of mitochondrial DNA already present, it does not need to be cycled as long as nuclear DNA. Because of the similarity of mitochondrial DNA, the bands resulting from gel electrophoresis will appear to be the same. Samples may be submitted to the Sequencing Service  of the Dolan DNA Learning Center, which can generate sequences and post them on the internet.

Results

From our results, we saw that the band width and positions are nearly identical to the human eye. This is understandable, as all human beings on the planet today share 99.9% of their DNA. The similarity allows us to trace our evolution and understand how we compare to each other, as well as to other species.

The next step in this lab will be sending our samples in to be sequenced, so we can compare our DNA in greater detail.

"Disease" Gene Testing

Background

DNA testing has multiple purposes, and is used in the study of evolution, as well as both forensic and paternal tests. One of its most valuable applications is checking for genetic diseases. Through DNA testing, one can test whether an individual is at risk for inheriting an illness such as cystic fibrosis or sickle cell anemia. Scientists take a sample of cells, amplify them using PCR, and analyze them through gel electrophoresis. In this lab, we will use the above steps to test for a gene (which is actually an intron. This is due to the ethical problems of testing for genetic diseases in class).

Procedure

Day 1: Collecting a Sample
A sample of skin cells is taken from the subject by chewing gently on the insides of the cheeks and then rinsing with a saline solution. The sample, once collected in a test tube, is spun in a centrifuge to clump all the cells together. The test tube is then immersed in two hot water baths, one at 56°C and one at 95°C. This is done to break the cell membrane and release the DNA. Once loose in the cytoplasm, however, DNAse would quickly destroy the sample DNA. This is why Instagene Matrix beads are added to the sample before it is immersed in the hot water baths. The test tube is then stored in a refrigerator until the next day.
Day 2: Polymerase Chain Reaction (PCR) Amplification

The sample of cells taken from an individual's cheek is much too small to be visible using gel electrophoresis (discussed during Day 3). PCR is therefore necessary in order to increase the size of the DNA sample before gel electrophoresis. In PCR, a primer is added to the solution. This primer acts like a heat-seeking missile, searching for the target gene sequence. The sample is then placed in a thermal cycler, which cycles through three temperatures- one to denature, or "unzip" the DNA, one to anneal to (find) their complementary sequences of DNA, and one to make new nucleotides attach to their complements on the unzipped strands. These three steps constitute one cycle; one PCR amplification consists of 40 of these cycles.
Day 3: Gel Electrophoresis
The bands of DNA are separated by size during gel electrophoresis, with the smaller bands being further towards the bottom (having progressed faster than the larger ones). There are three possible results.

If the gene being tested for is present in both chromosomes, a band of DNA strands 941 base pairs long will form. If it is present in neither, a band of DNA strands 641 base pairs long will form. If the gene is present in one chromosome but not the other, one band of each will form.

Results

There was a major source of human error for our result, which was that the gel was punctured by the pipette tip one two or three wells and somehow all of the samples bled through to some extent. All of our control samples were barely visible viewed in the correct light, as well as Lizzie's results (she was heterozygous, meaning she carried the 'disease' but did not herself show symptoms). Over half the class's results were homozygous negative, meaning they had the disease, so although our other test samples did not yield results, it would not be unreasonable to hypothesize that at least two of our tablemembers tested homozygous negative.