Unsuccessful PCR Genotypic Identification of the A4V Mutation on the SOD1 Gene using Human S9 cells
Abstract
The A4V mutation in the SOD1 gene is the most common mutation that causes amyotrophic lateral sclerosis (ALS). This missense mutation of cytosine to thymine on the 162nd base pair of the SOD1 gene changes the amino acid alanine to valine (Rowland and Shneider, 2001). To identify this specific mutation, a diagnostic assay was designed and tested using polymerase chain reaction (PCR) thermocycling and gel electrophoresis. Primers were created, utilizing the Yaku method of primer design, with an intentional adenine to cytosine mismatch located three base pairs away from the 3’ end of each primer. Previous experiments showed that this intentional mismatch would prevent the elongation of non-complementary sequences, increase specificity of the results, and decrease non-specific binding (Yaku et al, 2008). The hypothesis stated that the designed primers with the intentional mismatch would accurately detect the A4V mutation where present. The amplified DNA strands were analyzed by gel electrophoresis. Predicted lengths of amplified DNA sequence were 631 base pairs long when the two forward primers (mutant and wild-type) anneal to the complementary DNA strand (Patel and Loeb, 2001). To construct mutant DNA a site-directed mutagenesis method was implemented to place the A4V mutation into the wild-type DNA strand and amplify this transformed DNA to use against the mutant primers. The results of the designed primers against their complimentary DNA strand were inconclusive. In an attempt to understand the effects of ALS on people a sociological experiment was done to experience living with the symptoms of reduced mobility and analyze the physical, psychological and sociological effects. Using two disability index surveys taken each day and arm movement measurements, the hypothesis stated that as mobility decreased each day, its effect on everyday activities would increase. The R squared value of .9227 and p-value of .0001 prove to be statistically significant and support our hypothesis. This experiment is significant in improving the diagnosis of the A4V mutation and broadening peoples’ understanding of ALS and its effects on people.