Introduction
Amyotrophic lateral sclerosis (ALS) is a motor neuron disease that leads to progressive degeneration of nerve cells in the brain and spinal cord that control voluntary muscle movement. Physical symptoms include progressive muscle weakening in both upper and lower motor neuron regions that ultimately leads to death of 1 in 1000 people (Mitchell and Borasio, 2007). ALS is a dominantly inherited trait that lies on the SOD1 gene (Rowland and Shneider, 2001). It is more commonly seen as a sporadic disease rather than hereditary. Only 10% of ALS patients have a familial genetic mutation (Rowland and Shneider, 2001).
Twenty percent of the hereditary cases of ALS are caused by mutations on the SOD1 gene. There are over 90 mutations known on this gene, but the most common is the A4V mutation (Rowland and Shneider, 2001). The A4V mutation is strongly linked with familial ALS. Thirty eight percent of the families studied were found to have the A4V mutation (Rosen et al, 1994). This missense mutation in exon 1 constitutes a cytosine (C) to thymine (T) nucleotide substitution at the 162nd base pair (bp) that causes a change from the amino acid alanine to valine on codon 4.
Little is known about why mutations on the SOD1 gene lead to death of motor neurons in ALS patients (Collette and Rouleaur, 2002). An excess amount of glutamate metabolism in ALS patients could play a postulate role in the degenerative process (Rothstein and Martin, 1992). Glutamate is an amino acid that transmits messages between the axon and dendrites of motor neurons. The failure of this amino acid to be removed from the synaptic cleft is the result of a deficient cell membrane transport protein (Rothstein and Martin, 1992). The inhibition of glutamate transport is toxic to motor neurons due to the persistent elevation of its concentration in the synaptic cleft (Rothstein and Martin, 1992). The only medication approved for the disease is Riluzole and there are no known cures for ALS (Rowland and Shneider, 2001).
Our team developed a Polymerase chain reaction (PCR) based diagnostic assay to detect the A4V mutation. PCR amplifies a specific section of DNA based on where primers initially bind to a DNA template. There are three phases in each cycle of PCR that duplicate and amplify the section of DNA. The first is a denaturing step that occurs at 72°C that breaks apart the nucleotide’s hydrogen bonds, leaving two complementary DNA strands. The temperature is then lowered to allow the designed primers to anneal to the DNA template in their proper places. During the final phase of PCR, elongation, the temperature is raised to about 72°C in order for Taq polymerase to extend the primers and duplicate that section of DNA. These steps are repeated many times in order to amplify the section of DNA determined by the primers (Patel and Loeb, 2001).
Three primers were designed; a mutant forward primer to amplify the section of DNA with the A4V mutation, a wild-type forward primer to amplify the same section of DNA but without the mutation, and a reverse primer. To increase the specificity of our results we utilized the Yaku method in our primer design. Each forward primer contained an intentional mismatch located three base pairs away from the 3’ end. It was hypothesized that complementary DNA and primer sequences will elongate because one mismatch is not enough stop the Taq polymerase. This method decreases false-positive results because if two non-complementary DNA and primer sequences tried to elongate, the Taq polymerase would stop due to the intentional mismatch and the mismatch at the mutation. Gel electrophoresis was used to analyze the amplified DNA. Due to electrical charges in the gel the net negative charge from the spinal phosphate group in the DNA will cause the DNA to run from the negative end of the gel to the positive end (Raymond and Raymond, 1961).
DNA from S9 human epithelial cells was purified using the “Generation Capture Column Kit” created by Qiagen Inc. Based on the range specified by the Qiagen manual for using a 200 μl sample containing 5-7 x 10^6 cells/ml, the genomic purification should produce a DNA yield between 3-8 μg, (Qiagen Inc., 2007). The predicted annealing temperatures for the designed primers are 56.31 degrees Celsius (Forward Wild-type) and 54.36 degrees Celsius (Forward Mutant and Reverse) (Rychlik and Rhoads, 1989). After performing PCR using our designed primers, we predict our amplified DNA strand will show up at the 631 bp section (about 2/3 of the way down on the 1 kb plus ladder) of the gel (Raymond and Raymond, 1961).
ALS patients suffer from degenerative muscle function and often lose function of their muscles completely. To experience living with reduced mobility, the effects of various restraints on everyday activities was measured. This included limiting movement by attaching different weights to our arms that progressively got heavier as the week went on. The last two days of the week all mobility was restricted to only being in a wheelchair. The Roland Disability Index and Stanford Disability Assessment Questionnaire used each day to measure the degree of disability and how they affected every day activities. The the amount of arm lifts per min each day was also measured to show the progressive decrease in mobility that is caused by the disease. By measuring the amount of reduced mobility and how it effects a persons every day activities we predict that this experiment will produce results that show a negative effect of having the disease, such as decreased motor ability by signifying the amount of mobility lost due to the constraints along with the life costs that expand to the family of patients with ALS (Cleveland and Rothstein, 2001).