MMG Final Paper
Abstract
Written by: A43245931
Revised by: A43312212
Finalized by: A43220489
We hypothesize that by using PCR and Yaku-Bonczyk primer design methods we will be able to create a diagnostic assay to test for Angelman Syndrome by having an intentional mismatch on the 3rd base pair of both designed forward primers to reduce the likelihood of getting false positives by providing the possibility of two mismatches leading to a lack of annealing and extension. The intentional mismatch on the 3rd base pair will not allow for extension if using wild type DNA and mutant primers (or mutant DNA and wild type primers) because of the mismatches on both the first and third base pairs, so no bands would appear in the gel that is run. On the other hand, the intentional mismatch should not cause problems if using wild type DNA and wild type primers, the predicted band length for the designed primers is 800 base pairs. For our control primers the band length is 300 base pairs (Chotai, 1998). In the first genome prep, ratios of 1.435, 1.82, and 3.706, this means that pure DNA was extracted because two of the ratios are higher than 1.6. For our sociological experiment, a t-shirt was worn stating “I have Angelman Syndrome” and symptoms were replicated. As we predicted, the off-campus sessions yielded a higher number of negative responses and fewer questions were asked than the on-campus sessions.
Discussion
Written by: A43312212
Revised by: A43245931
Finalized by: A43112877
Angelman syndrome (AS) is a genetic disorder that mostly affects the nervous system. AS is a classic example of genomic imprinting because it is usually caused by a deletion or inactivation of genes on the maternally inherited chromosome 15, while the paternal copy, which may be of normal sequence, is imprinted and therefore inactivated (Dagli et al, 1998). AS results from the absence of a functional copy of the UBE3A gene inherited from the mother (Cross et al, 2007). UBE3A functions as both an E3 ligase in the ubiquitin proteasome pathway and as a transcriptional co-activator. This enzyme is involved in targeting proteins to be broken down within cells (Dagli et al, 1998). AS is expressed within the chromosomal region 15q11-q13 and the UBE3A mutation is located in the 602nd amino acid (Cross et al, 2007).
Primer Design
We hypothesize that we will be able to create a diagnostic assay by using PCR and Yaku-primer design methods to test for Angleman Syndrome by having an intentional mismatch on the 3rd base pair of both forward primers to reduce the likelihood of getting false positives by providing the possibility of two mismatches causing no annealing and no extension. The intentional mismatch on the 3rd base pair will not allow for extension if using wild type DNA and mutant primers (or mutant DNA and wild type primers) because of the mismatches on both the first and third base pairs, so no bands would appear in the gel that is run. On the other hand, the intentional mismatch should not cause problems if using wild type DNA and wild type primers, the predicted band length is 800.
Genome Preparation and Polymerase Chain Reaction
In order to show the presence or absence of the UBE3A gene mutation, we amplified a section of DNA of human epithelial cells from a normal patient through an allele specific PCR. The length of the amplified DNA was interpreted through gel electrophoresis to show the presence or absence of the UBE3A gene deletion. For our control primers, the amplified strand of DNA is 300 base pairs long, and our designed primers are 800 base pairs long. The control primers that were used are C1 and C2 (Chotai, 1998), they amplify exon 4 of the CFTR gene, thus it is a control because it does not amplify a portion of the UBE3A gene. The 2F primer and 3F primer, which are our designed primers, will be used in order to replicate the Yaku-Bonczyk Primer design method (Table 1). This is done by creating an intentional single base pair mismatch to ensure that annealing and extension will not occur if the DNA (mutant or wild-type) and primers (mutant-binding or wild-type-binding) do not match because if they do not match there will be a two base pair mismatch. A wild type genotype shows a band of 800 base pairs when using primers 2F and 2R are used. 2F is the designed wild type primer, while 3F is the designed mutant primer and 2R is the reverse primer. We also expected that no band would be present when primers 3F and 2R are used because mutant DNA is unavailable and the mutant primer is predicted to not bind to wild type DNA. If band is present in 2F primer it indicates a negative test therefore, AS is not present. We were able to show this because our successful designed wild type primers PCR showed that when wild type primers were used with wild type DNA, a band of 800 BP was shown. A facilitated conclusion was reached due to similar studies done on this syndrome which included the location of the deletion therefore guiding our predictions (Horsthemke et al, 2003).
In order to get the PCR assay to get function successfully, the amounts of substances in the PCR cocktail along with temperatures were varied. We successfully predicted that our mutant primers would be unsuccessful because of the absence of mutant DNA. For our designed wild type primers, we found the successful annealing temperature to be 46°C. We also were able to replicate our controls (Chotai, 1998) to get a band of 300 BP when run at 55°C, as expected.
Sociological Experiment
For our sociological experiment, we attempted to replicate, to an extent, the lives of the patients with AS, we wore shirts that say “I have Angelman Syndrome”. In our attempt to resemble an individual with AS, we mimicked some symptoms of AS, then we observed both students on MSU’s campus and people off campus and tallied reactions that were observed, including negative and positive responses towards us, resembling AS patients. In order to tally the responses, we counted how many people responded negatively towards the symptoms and in different sessions, how many responded towards wearing just the shirt, which was our control. Any look or phrase that was out of the ordinary was what was considered a negative response. In order to see how many people were genuinely interested in the disease, we also counted how many people asked us questions about AS while wearing the shirt. We hypothesized that those on MSU’s campus would be more accommodating and not give as many negative responses and ask more questions than those in the Lansing area, and that in both areas wearing just the shirt and not replicating the symptoms would yield fewer negative responses than wearing the shirt and replicating the symptoms. Our results (Figure 6) show that our hypothesis was supported because more questions were asked and fewer negative responses were shown on MSU’s campus.
Figure 1.
Successful PCR and gel electrophoresis for designed wild-type primers at 46°C for annealing. This figure shows PCR was successful at 46°C PCR was run with 94°C denaturation, either 46°C, 48°C, 51°C annealing, and 72°C for extension. Each sample was run in a thermocycler for 30 cycles. Using a semi-log plot (Figure 5), the bands in wells 2 and 4 were calculated to be around 1000 base pairs long, which was slightly higher than the expected length of 800 base pairs. This was due in part to the error in measuring specific bands of the ladder because the ladder did not spread far enough into the gel. Well 3 showed no bands because there was an error in the PCR cocktail. Bands did not show up in wells 5-8 because the temperature was too high for the primers to anneal to the DNA template, not allowing amplification to occur.
http://www.youtube.com/watch?v=E7s_KQfImdM