Performing Allele Specific PCR on Human Epithetical Cells to Identify

the Presence of the H1069Q Mutation on the ATP7B Gene Known to Cause Wilson’s Disease

By: TJ Morton, Kierstin Sanch,

Lauren Reinhold & Jenna Bursley

LB 145 - Cell and Molecular Biology

Eric Gurzell, Tim Oja, and Rachael Rinaldi

April 23rd, 2013

Sociological Experiment:

http://www.youtube.com/watch?v=NXqibKlBD6E

Abstract:

In this study, PCR and allele specific primers were used to identify the presence, or lack thereof, of the H1069Q mutation of the ATP7B gene of chromosome 13 in human cells, suggested to cause a rare disorder known as Wilson’s Disease. Primers were designed to specifically anneal to either mutant or wild-type DNA and amplify the desired segment of DNA in combination with a single reverse primer through PCR. The resulting product was hypothesized to be 540 base pairs for both mutant and wild-type DNA, though each primer would anneal specifically to its intended segment. This came as a result of the base pair mismatches detailed by the Yaku-Bonzyck primer design method, which prevents binding of bases, and thus elongation of desired DNA segments (Yaku et al, 2008). The product was calculated using the length of the primers individually and the distance between them, constituting the size of the desired segment to be amplified (Chenevert et al, 2013) The predicted product remains true in both wild-type and mutant hypotheses due to the unchanging length of the forward primer.  Through use of previously published primers, a product of 263 base pairs was expected. PCR was used to amplify the desired segment of DNA and gel electrophoresis was then used to analyze the resulting product. PCR using a wild-type, Cystic Fibrosis DNA template with a published forward primer (FPr1) and a published reverse primer (PRPr) across a melting temperature gradient yielded products that were 269, 269, 244, 244, and 222 base pairs long and approximately 570 base pairs using designed primers. As expected, PCR performed with the mutant forward primer (PFPr2), PRPr, and wild-type DNA yielded no amplified product. It is possible that future cases of WD could be treated through genetic therapy in order to minimize the effects of the disorder. Some sociological experiences of an individual with WD were emulated through diet modification and mimicking of speech disorders. Studies performed within the team and in the public allowed for recreation of WD symptoms and insight into the everyday life of an individual with WD.

Discussion:

The goal of this study was to perfect a set of specifically designed DNA primers and PCR protocol that could be used to confirm or deny the presence of the H1069Q mutation on the ATP7B gene causing Wilson’s disease in mutated and wild type human cells.

Wilson’s disease is an autosomal recessive genetic disorder in which copper is accumulated in the body’s tissues (Roberts and Schilsky, 2008). Typically this accumulation begins in the liver or in the nervous system and then continues throughout the body (Roberts and Schilsky, 2008). The disease is caused by a mutation of the ATP7B gene, which codes for the enzyme copper-transporting ATPase-2. More specifically it disrupts an ATP binding site on the SEHPL, a metal binding, motif (Shah et al., 1997). A motif is a significant, recurring sequence (Kunihiko et al, 1998)(Shah et al, 1997). In this case it affects the ability to bind to copper; and the malfunction of this enzyme prevents copper from being transported from the liver into the ceruloplasmin, where copper is usually properly circulated throughout the blood, or bile, and is where copper is released from the body (Shah et al, 1997).

The research team completed a preliminary experiment to gain experience with PCR and gel electrophoresis with E. coli and lambda primers and DNA. The team successfully performed PCR to amplify the target sequence of about 500bp for the lambda virus using lambda forward and reverse primers. Although the results showed some smearing, they demonstrate that the team is capable of properly performing a PCR assay and gel electrophoresis.

It was hypothesized that the Yaku-Bonczyk method used to design the primers will allow only the mutant forward primer (Q18A) to bind to the mutant DNA strand and only allow the wild-type forward primer (H18C) to bind to the wild-type or non-mutant DNA strand. It was predicted that this will occur as planned because of uncomplimentary mismatches on the third base pairs in, which will not anneal to either DNA type and cause kinks in the forward primers, which prevents the binding of Taq to the primer for that base pair, thus stopping the elongation of an unwanted sequence (Yaku et al., 2008). The reverse primer, R20, was designed to bind to both the wild-type and mutant DNA.

The first phase of this study consisted of using PCR with previously published primers and primers designed by the research team with wild-type human DNA to amplify a target sequence of DNA which contained the mutation of interest, H1069Q. The wild-type DNA was a control because it does not contain the mutation and thus the mutant primers will not anneal. The published primers, PFPr1, PFPr2, and PRPr, all served as controls to demonstrate that the team could properly perform PCR and gel electrophoresis, and also to determine that the mutation was or was not present in the wild-type/mutant DNA. A DNA ladder also worked as a control to analyze whether the primers effectively annealed to the expected sequence to amplify the target sequence. The original annealing temperature for the designed primers was predicted to be around 62ºC-64ºC, as seen in the methods. Further research into a study on identifying mutations on the ATP7B gene determined that a more accurate annealing temperature would be 55ºC (Waldenstrom et al., 1996). The predicted results for the PCR assay with designed primers included the annealing temperature at 55°C, the denaturation temperature at 94ºC, and the elongation temperature at 72ºC, which would produce a single bright band that corresponds to the length of the target sequence, 540 base pairs. The actual results show the proper PCR assay conditions for these primers were denaturation for 30s at 95ºC, annealing for 30s at a gradient between 54ºC-65ºC, and elongation for 45s at 72ºC, which produced single bright bands all at about 574bp. Because the primers amplified products very close to the target sequence on the wild type DNA, it was proven that the Yaku Bonzyk method was a success for designing the wild type forward primer, H18C, and reverse primer, R20. This denied the presence of the H1069Q mutation in the wild type DNA. For the published primers, the predicted results included a 95ºC denaturation temperature, 54ºC annealing tempurating, and a 72ºC elongation temperature, which would produce a single bright band that corresponds to the length of the target sequence, 263 base pairs (Iacob et al., 2012) (Witt and Landt, 2001). The actual results show the proper PCR assay conditions for these primers were denaturation for 10s at 98ºC, annealing for 20s between a gradient of 55ºC-60ºC, and elongation for 30s at 72ºC, which produced single bright bands at 269bp, 269bp, 244bp, 222bp, and 244bp. Because the published wild type primer, PFPr1, and the published reverse primer, PRPr, amplified products very close to the target sequence, the team was able to prove that the DNA was absent of the H1069Q mutation and the team was able to properly perform PCR. As predicted, when the published and designed mutant primers were both ran in PCR under the same conditions with the wild-type DNA no bands appeared, proving that the H1069Q mutation was not present.

The final portion of this experiment was conducted as a way of testing the social issues that affect a person with Wilson’s disease. The first social experiment was conducted with the research team cutting out all foods with high copper content in their diet for 30 days in order to see how this diet would factor into a WD patient’s life. The team found that foods like nuts and chocolate, which have notoriously high copper contents, were difficult to avoid because they are ingredients in many foods. By inserting cotton balls into their oral cavities, the team also attempted to mimic speech disorders, which occur as common symptoms of WD because of the copper build up in the muscles of the neck. (Kumar and Moses, 2005). Members of the team completed the Liebowitz Social Anxiety Test each week for four weeks to determine their anxiety levels caused by the disorder. It was hypothesized that by submerging themselves in everyday issues a patient with WD would typically encounter, the team would have an increased level of social anxiety. With an overall mean score for the team of 32.38 on this test, which diagnosed the team with moderate anxiety, this hypothesis was verified. Also, volunteers from the public were asked to perform a short tongue twister with cotton balls inserted in their oral cavities, then asked to rate the difficulty of this experience on a scale from 1-10. An average of 6.15 was slightly lower than the team’s initial expectations. Also, 80% of participants reported a sense of discomfort, stress, or embarrassment regarding their temporary speech impediment, again demonstrating the difficulties faced by a WD patient.

Errors in the experiment caused smears and non-specific binding to appear in the team’s gels after being ran, instead of having a clear band, which is what is expected. These could have occurred for a variety of reasons: the PCR assay lacking specificity, low annealing temperatures, causing non-specific amplification of the DNA sequence, an improperly made and poured gel, impurities in the sample DNA, degrading of the DNA, or an overloading of the wells (Cleland et al, 2012)(Roux, 2003). The team used a very basic method for calculating annealing temperatures, which could have caused some of these errors. This could be approached by using an equation to calculate the temperatures with the salt concentrations included. These new formulas can also aid in the explanation for the divalent cations and their effects that are present within the buffers used (Koressar and Remm, 2007). Also, while analyzing each gel, the team could have made a slight measurement error, causing the band lengths to be less precise. During the sociological section of the experiment, other errors arose such as: the way in which the volunteers understood the questions asked during the surveys, the team’s own personal biases, and the team removing cotton balls in order to complete certain activities without the speech impediment.

Future Directions:

Because the second phase of the experiment was not completed due to the team being unable to obtain Wilson ’s disease DNA. Once the team acquires this DNA, they will deal with testing both of the forward (H18C and Q18A) and the reverse primers (R20) on extracted DNA from mutated cell. If the results of the experiment showed that the mutated cell had the selected mutation, H1069Q, causing Wilson’s disease, the evidence would also appear as a single band 540 base pairs in length on the agarose gel when the mutant published and designed primers were used (Chenevert et al, 2013). This single band’s position or distance traveled corresponded to the number of base pairs in the target sequence it was believed to possess and therefore it was based on the designed primer binding locations (Shah et al, 1997). If this experiment and research were to be continued, the team would rerun PCR of the wild-type designed and published primers with higher annealing temperatures to rid the results of smears and non-specific binding. The use of restriction enzymes could also be used as a control to analyze if the predicted band in the agarose gel is truly from the desired sequence. Restriction endonucleases or enzymes are enzymes that cleave DNA at specific nucleotide sequences, which are known as restriction sites (Freeman, 2011). By using these enzymes the team could analyze the known base pair lengths that the enzymes would cut and then compare them to the base pair lengths of the amplified DNA; after degradation if the two lengths are the same, the team could then conclude that the predicted sequence of DNA had been amplified correctly (Roberts and Murray, 1976).

Figure:

Figure 4. PCR amplification of a segment of the ATP7B gene from wild-type DNA with designed primer set at an annealing temperature gradient of 56°C-65°C. PCR thermocycling conditions included an initial 1min denaturation at 95°C; 35 cycles of 30s denaturation at 95°C, 30s annealing at a gradient of 56°C-65°C, and 45s extension at 72°C; and a final extension for 10min at 72°C. After PCR amplification, gel electrophoresis was conducted in a 2% LB gel run at 130V for 30 minutes to detect amplified regions of DNA. Lane 1 contains 2μL of ThermoScientific GeneRuler 100bp DNA Ladder. Lanes 2-6 contain 10μL of a mixture of 9μL 1X PCR Buffer concentration of the designed wild-type primer PCR cocktail containing 40μL of nucleus free H₂O, 5μL of 10X PCR buffer, 1μL of 10mm DNTPs, 1μL of 100μm forward primer (H18C), 1μL of 100μm reverse primer (R20), 1μL of wild type genomic DNA, and 1μL TAQ polymerase; and 2μL of 6X Gel Stain. Lanes 2-6 show bands between 500 and 600bp. The bp length was determined by comparing the band amplified with the ladder using the graph. Each point on the graph depicts the specific distance travelled by each band of the 100bp Ladder with respect to its well. An R² value of 0.97556 was obtained for the logarithmic trend line of the plotted distance travelled. The equation obtained from the trend line was used to calculate the travelled distance of the bands in lanes 2-6 to get more accurate band size values. The values determined for lanes 2-6 were 574bp. All lanes show smearing and some non-specific binding. From this test it was determined that an annealing temperature above 58°C is optimal for our designed primers.