"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.