Team Ravioli's ALS Project

            Heritable amyotrophic lateral sclerosis (ALS) is caused by the Ala4Val (A4V) mutation on the SOD1 gene (Rosen, 1994). In order to successfully amplify the target DNA, or the mutation, we have designed primers to perform PCR on purified wild type and mutated DNA. If the designed primers are successful, the targeted A4V mutation will amplify to the mutated and wild-type of human epithelial CF DNA. Each primer we designed is twenty-one base pairs long; due to the length and location in proximity to the 3’ end (Brandes et. Al, 2007), we predict that the Taq Polymerase will bind to the DNA and our primers will be successful at duplicating the DNA because the primer has a better chance of annealing if the mutation is placed closer to the 3’ end (Yaku et al. 2008). We assume that Primer M with Primer R will anneal to amplify the A4V mutation through PCR in the purified DNA. Purified DNA is needed to obtain good, quality results from our primers, therefore Genomic Extraction will be used in order to purify the DNA that will be used. We expect to obtain approximately 1.07x10^5 fragments that are 1,156 base pairs long of our target DNA through 30 cycles of PCR. In order to gain full knowledge of the impact of ALS on the human body we also performed a 30 days experiment in which we reenacted the symptoms of ALS over a 30 days period and recorded our results. We expected that the 30 days experiment would give us much insight as to how difficult ALS is to live with, which it did.  Our goal was to impact not only us but also additional scientists following our research.

Experiment Summary
            Amyotrophic lateral sclerosis (ALS), which is also known as "Lou Gehrig's Disease," is a neurodegenerative disease in which the nerve cells in the spinal cord and brain are affected and progressively shut down (Deng et al., 1995). The disease is very rapid and most often leads to total paralysis before death, which typically occurs within two to five years after diagnosis (Gurnery et al., 1994).
           The mutation, ALA4VAL (A4V), on the SOD1 gene on chromosome 21 has been linked to ALS as a cause for about 20% of cases (Gurnery et al., 1994). Through PCR we are trying to amplify the A4V mutation. In order to successfully amplify the target DNA, or the mutation, we have designed primers to perform PCR on purified wild type and mutated DNA. Therefore, it was predicted that by using designed primers targeted at this mutation, that we would be able to successfully test for the ALS gene using PCR with an annealing temperature of 54°C producing a fragment of 1169 bp. At the completion of this experiment we not only wanted to find if our designed primers worked, but also to understand what it would be like to have the disease itself through a thirty-day project designed to simulate ALS patient’s symptoms and progress through stages that we predict will lead to a major decrease in an individual’s satisfaction with life (Maessen et al, 2010).

Designed Primers

Primer sequences were created to make fragmental copies of DNA strands. These primers work by binding to the target areas, where the mutation is at, and annealing to them in order to make copies of the strand sequences (Yaku et al., 2008).  The first forward (Primer W) primer designed contains the wild-type genomic sequence and the second forward primer (Primer M) contains the mutated genomic sequence. The primers were created with the mutation closer to the 3’ end of the sequence because it allows for a better chance of the codon attaching to the DNA (Brandes et al., 2007). The designed primers are 21 base pairs in length and will work because primers should be anywhere from 15 to 30 base pairs for successful PCR because it is long enough to not have a duplicate sequence anywhere else on its exon (Yaku et al., 2008). We have predicted that due to the design and length of our primers that they will be successful at polymerizing and binding with the DNA (Brandes et al., 2007). The significance of these primers being successful is astronomical in further understanding how ALS attacks the human body and discovering potential gene therapy solutions.  However, if we do not have successful primers, they will not anneal to the target DNA and the taq polymerase will not be able to synthesize the DNA (Yaku et al., 2013).  Unfortunately, under the time constraints, we were unable to test our designed primers via PCR in the laboratory; therefore, at this moment the success of our designed primers is uncertain and waits further experimental testing.

Experimental Findings

DNA provided by Dr. Douglas Luckie from epithelial cells of patients with Cystic Fibrosis acts as a control. DNA Purification is essential to PCR because without a concentrated samples of DNA, PCR would be unsuccessful. In the Qiagen handbook for effective genomic DNA purification, the purification of DNA from other components has four steps: disruption, lysis, removal of protein and contaminants, and the recovery of the DNA. We predicted that this would yield about 5-30 µg of DNA because that is the average yield obtained when using 20 mg of tissue homogenates according to Qiagen.  Using the Epoch Micro-Volume Spectrophotometer, the DNA concentration was found to be 1.054 in the purified solution.  However, when DNA collected from capture column was added into proven PCR cocktails via Lambda control along with successful published primers, the desired fragments of 215 bp were not replicated along a gradient of temperatures (44.2°C, 41.3°C, 38.9°C, 36.8°C, and 35°C).  Initial troubleshooting resulted in the creation of five cocktails, in which the concentration of primers, taq polymerase, dNTPs, DNA, and dNTPs and primers were doubled respectively in each cocktail and run through PCR with an annealing temperature of 40°C.  Early assessment of gel electrophoresis mislead us to the belief that the cocktail containing double primers along with the cocktail containing double DNA had generated fragments of 215 bp in length.  However, further analysis of both cocktails run along a gradient of 44.2°C, 41.3°C, 38.9°C, 36.8°C, and 35°C demonstrated that the bands were merely a result of primer dimmer with a bp length of less than 100.  This revelation led to the belief that the genomic preparation of DNA was unsuccessful; therefore, a sample of DNA from Graduate Assistant Eric Gurzell was used as a control against our purified DNA with the original proven PCR cocktail from the Lambda control.  The resulting gel electrophoresis generated a targeted fragment at approximately 215 bp at an annealing temperature of 35°C; therefore demonstrating that genomic preparation of DNA was unsuccessful resulting in the inability to amplify the targeted DNA fragment.

30 Days in Life of a Patient with ALS
            ALS is a very difficult disease to live with. That being said, it was our goal to attempt to mimic the disease as best as we could in order to gain some insight as to exactly how difficult it is to live day to day with ALS. In order to replicate this we start by having slight “symptoms” in the first week of our experiment and continue to gradually increase the severity of these symptoms in an effort to mimic the duration of the entire disease into a 30-day timeline. The data was recorded using the satisfaction of life scale that rates quality of life (Abdel-Khalek, 2006). We also tested three additional every day tasks each week that would be affected by ALS: 1. How long it takes to take to button a shirt, 2. How long it takes to write the same paragraph, and 3. How long it takes to walk up the stair of Holmes Hall. Due to the severity of this disease we have predicted that by the last week when we are only able to eat through a straw and get around with assistance, our satisfaction of life will be at an all-time low. If the 30 days experiment is successful then we will be feeling extremely useless and that our life contains little importance to the world by the end of week 4 demonstrated by our score on the satisfaction of life scale which should be tremendously low based on collected data of ALS patients (Veldink et al, 2002). If the 30 days experiment is unsuccessful then we expected to not have much of a difference, if any at all, of what our control satisfaction of life level is compared to what our experimented satisfaction of life level is; also based on the SWLS (Diener et al., 1985).   Ultimately, the four tests performed provided a unique perspective of the psychological and sociological struggles that ALS patients endure.  Based on the SWLS, in contrast to control patients, subjects inflicted with ALS symptoms went from being “satisfied” with their lives to “extremely dissatisfied” in just four weeks.  Everyday tasks and abilities such walking up flight of stairs and buttoning up a shirt went from being taken for granite to tedious to impossible.  Inflicted subjects who were once outgoing became insecure, shy and avoided public places that they used thrive in.  This regression of “self-worth” is a common theme of ALS patients leading to rising numbers in foreign nations where euthanasia of those inflicted with ALS is legal (Carver and Foley, 2004).

Figure 4. Agarose gel of control primers with the original cocktail and foreign DNA, double the concentration of primers with foreign DNA, and triple the concentration of primers with domestic DNA. Thermocycling conditions included a 5-minute denaturing at 95ºC with 30 cycles of denaturing at 95ºC for 30s, annealing at 50ºC for 30s, and elongating at a gradient of temperatures between 35ºC and 44.2ºC for 60s. The final elongation phase lasted for 5 minutes at temperatures in the same gradient. The agarose gel was made with 1xTBE and run at 110 Volts for twenty-five minutes. PCR products using Primer E5F (5’-AGT GAT TAC TTG ACA GCC CA-3’) and Primer E5R (5’-TTC TAC AGC TAG CAG GAT AAC A-3’) were placed in five premade wells in the gel. A 1kb DNA ladder was placed in the first well to act as a control to determine the length and success of the PCR products. Targeted annealing position of the primers was located on the 13,803-14,018 base pairs on exon 5 of the SOD1 gene. The expected band size was 215bp. (A) The PCR cocktail was the same as that used in Figure 2, though DNA obtained from a foreign party (DNA B) was utilized. In Well 4, a possible successful band was observed, as it appears to be in line with the 215bp mark. (B) The PCR cocktail resembled that which was used in Figure 6A, but was run with DNA B. The assay was classified as unsuccessful due to non-specific binding and primer dimer. (C) The PCR cocktail was ran with the DNA analyzed in Figure 4 (DNA A), but triple the concentration of primer was utilized. In Well 4, where the annealing temperature was 35ºC, a possible successful band could be observed.