Title Page authored by: A44365838

Title Page revised by: A44774656

Title Page finalized by: A45297858

Genetic Detection of Angelman Syndrome Patients with the UBE3A Mutation in IB3 Cells Using PCR and Gel Electrophoresis

By: A44365838

A44774656

A45727337

A45297858

LB 145 Cell and Molecular Biology

Section 9-Mondays & Wednesdays 7:15-9:15pm

Professor Luckie

TA: Candace Igert

LAs: Zachary Gaudette and Kristin Elinski

Due: Monday, April 22, 2013

Abstract

Authored by: A44365838

Revised by: A44774656

Finalized by: A45297858

Angelman Syndrome (AS) is a genetic mental disorder. One mutation responsible for causing Angelman Syndrome is 856delG, which causes an overall change in the genotype and phenotype (Fang et al., 1999). Through PCR, a specific DNA sequence can be replicated numerous times and one can determine if a mutation is present in human DNA. Primers can bubble over base pair mismatches, causing non-specific binding. Thus, two different primers were used with distinctive mutation sites to avoid inaccurate results. This experiment was conducted to determine whether or not a primer with a mutation at the 3? end would differ from a primer with a mutation in the middle when annealing to a DNA strand. Previously published primers were used as a control to analyze the experimental process and account for human error. It was hypothesized that the primer with the mutation on the 3? end would not bind to the template DNA strand (Borodovsky and McIninch, 1993). However, it was hypothesized that the primer with the middle mutation would bind to the DNA, because it would ignore the presence of cytosine in the DNA strand (Fang et al., 1999). Experimental trials refuted our hypothesis and showed a base pair product of 367 for both the end mutation and middle mutation primers. The results illustrated that both designed primers were able to bind to wild-type DNA. In addition to performing a PCR diagnostic assay, the research team also simulated having Angelman Syndrome for 30 days. They recorded the observations of onlookers at specific sites to determine the perception of patients with AS. Through the enactment of AS, it was found that 92% of people viewed did not pay any special attention to the acting member. Accommodations available to individuals with AS on the Michigan State University campus were also recorded. Examining how primers ignore base pair mismatches and deletions can result in the detection of unknown diseases, which can lead to possible treatments or cures. Furthermore, the development of new primers specific for detecting the 856delG mutation could lead to more progression in the diagnosis of this rare disease.

Figure 4: Final PCR experiment conducted showing the amplification of several DNA sequences with various primers. Four PCR Cocktails were created, each containing the following: 40μl of nuclease-free water, 5μl of 10x PCR buffer, 1μl of 10mM dNTPs, 1μl of DNA template, and 1 μl of 5 U/μl Taq polymerase. After the generic ingredients were added: 1μl of middle mutation forward primer and 1μl of middle mutation reverse primer was added to the middle mutation microfuge tube, 1μl of end mutation forward primer and 1μl of end mutation reverse primer was added to the end mutation microfuge tube, 1μl of wild type forward primer and 1μl of wild type reverse primer was added to the wild type microfuge tube, and 1μl of published forward primer and 1μl of published reverse primer was added to the published microfuge tube. Each cocktail was placed in the thermocycler where denaturation was to occur at 95?C for 30 seconds. From there, a gradient was set in the thermocycler where each cocktail would be at its own separate temperature depending on the temperature it would best anneal at. The gradient for the annealing stage was from 37?C to 49?C. After annealing for 30 seconds, the temperature was raised to 72?C for 20 seconds. This completed one cycle, then 29 more cycles were performed. After the 30 total cycles, a final extension of 6 minutes was used. To test our PCR amplification, we ran gel electrophoresis and observed the products. In wells 1 and 8, a 1Kb+ ladder was pipetted to give a control for comparison. By measuring out the migration distance of the base pairs of the ladder, we were able to determine our actual product base pair size using the equation of the ladder curve. Lanes 2 and 3 contain middle mutation primers, lanes 4 and 5 contain end mutation primers, lane 6 contains wild type primers, and lane 7 contains control primers. After running the gel and observing it under UV light, we observed the following band lengths based on the 1Kb+ ladder: Middle Mutation Primer = 367 bp, End Mutation Primer = 367 bp, Control Primer = 652 bp, Wild type Primer = 1364 bp.

Discussion

Authored by: A44774656

Revised by: A44365838

Finalized by: A45727337

Experimental Summary

Angelman Syndrome is a rare neurobehavioral disorder that occurs in 1 out of every 20,000 individuals (Lossie et al, 2001). A patient with Angelman Syndrome experiences mental retardation, seizures, laughter, abnormal gait, and frequent smiling (Kishino et al, 1997). This disease is a result of defects in the maternal chromosome 15q11-13, which leads to large maternal deletions, imprinting mutations, uniparental paternal disomy (UPD), and loss of function mutations of E6-associated protein (E6-AP) ubiquitin-protein ligase (UBE3A) (Albrecht et al, 1997). Mutations, such as a deletion of Guanine at base pair 856, can lead to an abnormally short version of ubiquitin protein ligase E3A. This results in the symptoms associated with Angelman Syndrome (Clayton-Smith and Laan, 2003). In order to determine the presence of the 856delG mutation in the UBE3A gene of given human epithelial cells from a CF patient (IB3 cells), our research team performed a polymerase chain reaction (PCR) diagnostic assay.

Before completing our own PCR with designated mutant primers, we conducted PCR using Lambda virus DNA. This was done in order to ensure that the proper PCR procedure was followed accurately. Two different cocktail mixtures were created with DNA, PCR buffer, Taq polymerase, forward primer (5?GATGTATGAGCAGAGTCACCGCGAT-3?), reverse primer (5'-GAGGGTGAAATAATCCCGTTCAG-3'), dNTPs, and nuclease-free water. These cocktails were placed in the thermocycler and then observed under UV light. After completing our PCR assay with the Lambda virus, one of our controls, we extracted genomic DNA from IB3 cells. In addition to Lambda, we also used a water pipetting control. This enabled us to determine the accuracy of our pipetting technique during PCR (Figure 6). We extracted the DNA in order to eliminate proteins and other contaminants present (Figure 3). The purified DNA obtained (7μg) was used with our primers to conduct PCR.

Four different PCR tests were run to determine the presence of the 856delG mutation. In the first test, we used a mutant primer with the 856delG mutation in the middle and wild-type DNA. In the second test, we used mutant primers with the 856delG mutation in the end and wild-type DNA. In our third test, wild-type primers were used with wild-type DNA. In our fourth and final test, our control primers were used with wild-type DNA. Our research team decided to design two different mutated primers to determine whether the presence of the mutation impacted the way the primer was able to bind to the DNA template.

After completing these four tests, sociology and psychology were incorporated into a designed procedure involving experimental simulation. Each of the members of our research team enacted certain symptoms associated with Angelman Syndrome. This experiment was conducted in order to determine what a patient with Angelman syndrome must deal with on a regular basis. Images of each of the group members were taken as they enacted these symptoms, and a slideshow was then composed.

Original Predictions

It was predicted that using PCR and gel electrophoresis would enable us to determine if the deletion of Guanine in the UBE3A gene was present in the template DNA strand (Albrecht et al, 1997). For the first test, we predicted that the mutant primers with the middle mutation (MPMMs) would bind to the wild-type template DNA, because the forward primer (5?-GATAAAAAT*AACAAGAAAGGC-3?) would ignore the presence of Cytosine in the template DNA and continue to bind to the rest of the sequence (Figure 1). It was hypothesized that the forward MPMM would bind from base pair 847 to 867 and the reverse MPMM (3?- GAGTTCCTATCCACTATCGAGT-5?) from base pair 1170 to 1149, resulting in a 323 base pair product. Both of these primers were anticipated to anneal at a temperature of 46?C. For the second test, our research team predicted that the forward mutant primer with the end mutation (5?-GCTCTGAGATAAAAAT*A-3?) would not be able to bind to the wild-type DNA from base pair 840-856, resulting in no 330 base pair amplification product (Figure 1). Because of the presence of the mutation at the end of the primer, we thought that Taq polymerase would not be able to attach bases to the 3? end and elongate the template DNA strand. However, we believed that the reverse MPEM (3?-GAGTTCCTATCCACTATCGAGT-5?) would still anneal to the wild-type template DNA strand from base pair 1170-1149, because of the absence of the 856delG mutation from base pairs 1170-1149. The annealing temperature of the mutant primers with the end mutation was calculated to be 37?C.? For our third test, we predicted that both wild-type primers would anneal to the wild-type template DNA strand. We anticipated that the forward wild-type primer (5?-GATAAAAATGAACAAGAAAGG-3?) would bind from base pair 847 to 867 and the reverse wild-type primer (3?-GGAGTTCCTATCCACTATCGAG-5?) would bind from base pair 1170 to 1149, resulting in a 323 base pair amplified product (Figure 2). For our fourth and final test, we expected that the forward control primer (5?-AGGAGTTGTGGTAAATAGTGCA-3?) and the reverse control primer (3?-ATCTTCGTCTAGTATACGGTTT-5?) would not bind to the wild-type template DNA strand, due to the presence of Cytosine.

For our sociological experiment, we hypothesized that individuals walking by those simulating having Angelman Syndrome would mostly ignore those enacting the symptoms, due to the assimilation of people with mental disabilities into modern society (Park, et al., 2003). In addition, we hypothesized that the library would have the most accommodations including: ramps, handicap bathrooms, closed study rooms, and health clinics (Figure 5).?

* The deletion of Guanine occurs between Thymine and Adenine

Ultimate Findings

Our results show that the primers used with the Lambda DNA (see experimental summary) annealed to the template DNA strand and created a 415 base pair product (Figure 7). The 1kb plus ladder used allowed us to determine the exact base pair product size using a line of best fit (Figure 7). The Lambda DNA primers attached to the template strand, because the base pair sequence of the primers and the DNA complemented one another. When placed in the thermocycler, the three phases of PCR occurred successfully with the Lambda PCR diagnostic assay. Our results also show that for our first test, the designed mutated primers with the middle mutation (MPMM) annealed to the wild-type template DNA strand, resulting in a 367 base pair product (Figure 4). This refutes our original hypothesis, because even though we predicted these primers would anneal to the template DNA strand, we predicted that a 323 base pair product instead of a 367 base pair product would be produced. Our research team believes this occurred because of an error when using Primer-BLAST. For our second test, the results show that the mutated primers with the end mutation also annealed to the wild-type DNA strand and produced an amplification product of 367 base pairs (Figure 4). This refutes our original hypothesis, which was that the MPEMs would not be able to anneal to the template DNA strand due to the one base pair mismatch at the end of the forward primer. We believe this occurred because of the temperature gradient. The low temperatures used caused non-specific binding, and gave off an undesired base pair product. Our findings for our third test indicate that the wild-type primers annealed to the wild-type DNA strand and created a base pair product of 1,364 base pairs (Figure 4).? This, again, refutes our original hypothesis. We predicted that the primers would anneal; however, we believed that only a 323 base pair product would be produced. Our results for our fourth test indicate that the control primers annealed to the wild-type DNA and created a 652 base pair product (Figure 4). We predict that this occurred since the control primers annealed to the introns surrounding exon 8 (Fang et al., 1999).

For our sociological experiment, our results indicate that the library had the most accommodations (Figure 5). They also show that 96% of people did not notice the disabilities associated with Angelman Syndrome (Figure 5). This supports our original hypothesis, since we believed that people would ignore disabled individuals due to the large portion of encounters with disabled people in every day life.

An Angel from above

In addition to PCR, a 30-day experiment was conducted to analyze the symptoms associated with Angelman Syndrome. Our research team enacted out the following symptoms: hysteric laughter, trouble walking, trouble eating, and hyperactivity, in six different locations (Holmes Hall, McDonel Hall, Hubbard Hall, MSU Library, Meridian Mall, and Brody Hall). In addition to enacting the symptoms, we also took into account the various accommodations offered to patients with Angelman Syndrome at MSU. We counted the number of ramps, closed study rooms, health clinics, and handicap bathrooms in Holmes Hall, Hubbard Hall, the MSU library, Brody Hall, and McDonel Hall. Our results indicate that the library had the greatest number of accommodations (26)(Figure 5), and Brody Hall had the second highest number of accommodations (11)(Figure 5). Our goal in creating this experiment was to educate the public about the challenges a person with Angelman Syndrome faces in everyday life.

Future Directions

A total of 6 different PCR tests were conducted with our primers in order to get a variation of results. For the first 2 PCR tests, our bands for all of the primers appeared faint. We predicted that this was because of an oversaturation of the primers. A high primer concentration usually results in an accumulation of nonspecific product (Robertson and Walsh-Weller, 1998). Thus, we suspended our primers in 1600μl of distilled water. This created a 12.5 μM primer solution. After conducting PCR for the third time with our primers with the new primer concentration, we found that the primer concentration was not the problem. Our results showed that there were no amplification products produced for any of the four cocktail mixtures. We then realized that before suspending the primers in distilled water, we should have saved an adequate portion and placed it in a different tube. This would have enabled us to use our previous ordered primers if the suspended ones did not work.? Because of this error, we were not able to obtain results for the fourth and fifth PCR tests that we ran with the four cocktail mixtures. Through experimental error in the fifth test, our research team found that the SYBR Safe dye is only used when making the gel, and the loading dye is the only one added to the cocktail mixture. The SYBR Safe dye enables us to see the bands, because it fluoresces. The loading dye helps make the sample more viscous so it sinks into the wells of the gel. In addition, the loading dye stains the DNA fragments. The SYBR Safe cannot be added to the cocktail mixture, because it has a positive charge; thus, the bands will run towards the negative end instead of the positive end (Figure 8). We then performed our sixth PCR test the last day of lab, and we finally obtained results. By troubleshooting, we concluded that non-specific binding due to low temperatures created the large base pair sizes obtained for all of the primers used. When using low temperatures with primers during the annealing phase, the binding of the primer to a desired specific DNA sequence may not occur (Waterboer et al., 2006). This is because the primer can attach to a sequence that is more easily accessible, or bubble over additional base pairs (Waterboer et al., 2006). In addition to non-specific binding, we also believe that the large concentration of DNA used for our sixth test may have affected our results. A larger quantity of DNA enables the primers to anneal to the DNA strands more readily, which also results in non-specific binding (Waterboer et al., 2006). If we had more time to work on this experiment, we would make sure we were using a proper primer and DNA concentration.