Identification of the G1013R Homo Sapien Mutation within Marfan Syndrome Patients via Allele-Specific PCR
By: Bhavik Patel, Cole Scott, Susannah Haupt, and Caitie Behnke
LB 145 Cell and Molecular Biology
Tuesdays & Thursdays 7PM
TA: Eric Gurzell
LA?s: Tim Oja and Rachel Rinaldi
4/18/2013
(Title page written by: A44621342)
(Revised by: A45376783)
(Finalized by: A45328864)
Abstract
PCR is used to identify and study specific mutations. Thus, the purpose of this experiment was to see if PCR could accurately identify a Marfan Syndrome (MFS) genetic mutation. We hypothesized that two primers, a mutant type and reverse type, designed by the Yaku-Boncyzk method, would specifically anneal to the G1013R mutation through PCR while the wild type primer will not anneal to the mutation due to an intentional mismatch. The resulting agarose gel electrophoresis determined if the mutation was present in a patient by showing a band length of 1183 base pairs. Three primers were designed using the Yaku-Boncyzk method. Next, DNA purification was completed using a Qiagen Generation Capture Column Kit with isolated DNA from the cells of MFS and non-MFS patients. A PCR was then ran and analyzed using agarose gel electrophoresis. The team predicted that the G to A primer would attach to the mutant DNA because of a previous successful PCR (Nijbroek et al, 1995). While the team predicted this, no detectable band appeared at the 1183 base pair with the mutant primer. Instead primer dimers appeared which questioned the efficiency of the mutant primer and reverse primer. The primer dimers are significant because it is a problem many scientists experience in their own research. Marfans not Martians was a second study measuring social anxiety and pain levels in the life of a MFS patient. Each team member picked an emergency randomly that a MFS patient might encounter in their lifetime and lived with the symptoms for four days. This occurred each week for three weeks; each time a team member would get a different emergency. The social anxiety and pain factors were measured using the Liebowitz Social Anxiety Scale and Pain Comparative Scale from Packard?s Hospital. We found that there was no significant difference between pain and social anxiety.
Discussion
Experiment Summary
Marfan Syndrome (MFS), the most common connective tissue disorder, characterized by ocular and aortic challenges, is expressed in every 1 in 5,000 people (Milewicz et al., 2005). One mutation that causes this disease, G1013R, is caused by a substitution from G to A in the fibrillin-1 (FBN-1) gene on chromosome 15 (Nijbroek et al., 1995). This mutation results in the faulty synthesis of FBN-1 glycoproteins that form microfibrils and elastin fibers. The degradation of microfibrils that control transforming growth factors-β results in the overgrowth of bones, causing a degradation of elastin fibers in the extracellular matrix that produce the disease-characteristic difficulties in the eyes and aorta (Keane et al., 2008).
In 1991, Dietz et al. ran an experiment that successfully located the C1409S Marfan Syndrome mutation using PCR. Although their PCR worked for C1409S, we questioned whether or not a PCR run with a specific annealing temperature on the same FBN-1 gene could accurately diagnose a patient for Marfan Syndrome with a G1013R mutation. From this we hypothesized that because of the nature PCR, specifically designed primers will attach and amplify the G1013R mutation or wild type mutation on the fibrillin-1 gene; thus allowing analyzation of gel electrophoresis to determine if the DNA of a patient is sufficient enough to diagnose Marfan Syndrome.?
In addition to our genetic MFS experiment, a sociological experiment titled ?Marfans not Martians? was conducted. When designing our social experiment we hypothesized that the symptoms would cause social anxiety levels and pain levels felt would be equivalent for each symptom. MFS causes chronic pain and visibly physical attributes, such as elongated limbs, quite exaggerated from the average person. In a study by (Tongerloo et al.,1998), MFS patients versus a control group were studied on sociological issues. Of the MFS participants, 65% had issues with bullying and 53% had self-image issues. To add to this social strain, other manifestations involved in the syndrome, such as aortic aneurysm and scoliosis, cause social anxiety. Pre- and post-surgery braces are necessary for these symptoms as well as a head bandage wrap for post-ectopia lentis surgery. While mimicking these symptoms for three weeks, the group members rated their pain and annoyance using the Pain Comparative Scale from Lucile Packard?s Hospital, compared to the social anxiety levels using the Liebowitz Social Anxiety Test.
Original Predictions and Ultimate Findings
Lambda was first used as a control system that helped fix methodology errors in running PCR and gel electrophoresis so the actual mutant Marfan Syndrome DNA PCR and gel electrophoresis will not have any methodology errors. Our control will be using the WTD with wild type DNA reaction as far as PCR and primer binding goes, and the ladder for the agarose gel electrophoresis. Once a confirmed positive PCR from the published primers is completed, that will then be used as a control as well.
When ordering the designed primers certain precautions were taken to insure correct annealing, such as using Primer Blast. This was done by placing the Fibrilin-1 gene sequence into the program along with WTDf, and Dr primers. The MD primer was not found by Primer Blast due to the intentional mismatch from the Yaku-Bonyczk primer design method. The primers showed annealing that was needed to replicate wild type DNA with WTD primers and the mutated DNA would be replicated with MD primer plus Dr primer. Both the purified wild type and mutant type DNA were tested in the Epoch Spectrophotometer and concluded that both contained sufficient amounts of DNA. Wild type had a 1.73 260/280 which is a low purity ratio and mutant type had a 1.94 260/280 which is a higher purity ratio. This concludes that PCR can occur with the amounts of DNA used (Clark et al, 2000).
Troubleshooting became necessary when results were indefinable or nonexistent. Trouble shooting ideas were found in Optimization and Troubleshooting in PCR (Roux, 1995). To troubleshoot our designed PCR and control, we first changed temperatures for annealing. The original annealing temperatures for the designed wild type primer and mutant type primer was 65?C. The published PCR temperatures remained the same for control purposes. When temperature became 10 to 15?C lower than calculated, between 50-60?C, the team decided more troubleshooting should be done. Salt concentrations were then changed in all PCR reactions. This is because the salt in the PCR buffer, Mg+ ions, can affect several situations in the reaction. For example, annealing, specitivity of product, and the use of Taq polymerase can be affected (Innis et al, 1990). Originally using 5μL of PCR buffer, the group tested PCR with double salt concentration so that buffer was 10μL and a lower salt concentration lowering the buffer to 3μL per PCR reaction. Due to the low purity of wild type DNA, doubling the salt concentration was necessary to receive bright, accurate bands. On the other hand, due to the high purity of the mutant DNA and it being slightly over-concentrated, bands were received when using the normal salt concentration, while no bands appeared, aside from the primer dimers, when doubling the salt concentration.
Due to the change in buffer concentration, positive results became possible for the controlled PCR reactions and unspecific results for designed and mutant PCR reactions. As seen in Figure 2 the published primers for wild type annealed and produced a base pair length of 365 which should have been 356, while both mutant and wild type designed PCR showed unspecific binding at base pair lengths unlike what should have been produced, around 575 base pairs. All base pair lengths for this gel were configured by the equation produced in Figure 3. If these were to anneal and amplify correctly, the base pair length of both the mutant type PCR and wild type PCR should produce bands at 1183 base pairs.
In wild type PCR with high concentration and mutant PCR at high and low concentrations, a second band was seen at base pair length 110. This band shows nonspecific binding which was caused by low annealing temperatures. This causes annealing to happen quickly and accurately. In each lane, at 31bp, primer dimers appeared. In past research primer dimers have been found around 59bp and can be seen in each lane when it occurs (Warner et al, 1993). Once this was discovered, the group tested the design primers on NetPrimer. NetPrimer confirmed that the designed primers are able to anneal to one another causing primer dimers. An example of this phenomenon can be seen in Figure 4 showing which shows the base pairs of our designed primers will anneal to one another. Higher salt concentrations can also cause a higher ability for primer dimers to be formed (Innis et al, 1990).
For our sociological experiment, we predicted that the levels of sociological and physi??????cal pain due to the MFS symptoms, mimicked by the research team, would not differ. In Figure 5, the comparison graph shows that after the three weeks the group could conclude that social anxiety rose to be a higher problem than the physical pain, rejecting our original hypothesis. While pain is a major part of the life of a MFS patient, the sociological stressors should be noted.
Future Directions
Although we practiced the PCR and gel electrophoresis methods to eliminate errors in experimental methodology, there was still room for error that future research can eliminate. Due to the fact that MFS recognizes many mutations on the FBN-1 gene, making multiple mutant primers for multiple FBN-1 mutations would have yielded more accurate results (Nijbroek et al,1995). The further research increases the likelihood of finding bands for the mutant DNA at its specific base pair.
When testing primers, NetPrimer should be used to confirm primer dimers will not be produced. This would allow a higher chance for the correct replication of the mutation. The primers first designed should be replaced due to the issue with primer dimers. With time, Lambda PCR should be brought back for more control and comparison when running gel electrophoresis with mutant DNA PCR product.The amounts of PCR pipetted into the wells of the gel should be varying to find the optimal amount of PCR product that can be added to the well to still receive an accurate, bright band. Also, there should be a PCR cocktail run with mutant DNA and wild type primers to ensure that the bands are accurate and that every primer is not binding to both DNA types.
Concerning the sociological experiment, future research in social stress of persons with MFS should be studied. More symptoms could be expressed, with a longer extended period of expression. Another interesting factor that could be added to future research would be attending weekly on-campus and off-campus support groups. At these meetings, the researchers would explain what they went through regarding pain and social anxiety during the week. At the end of the experiment, other members of the support groups that were not on the research team would take a survey on the researchers, regarding how they perceived the researchers socially. This information would add a new level that would allow the social anxiety the researchers felt to be compared to the way the public views the researchers socially when they are expressing the symptoms of MFS.
Figures
Figure 2: Amplification of DNA segment containing G1013R mutation using designed primers and amplification of DNA wild type segment using published and designed primers, while varying salt concentration of PCR cocktails. Thermocycling conditions included an initial denaturation step held at 95?C for 2 minutes, followed by 30 cycles of 95?C for 30 seconds, various annealing temperatures all held for 30 seconds, and 72?C for 45 seconds. The optimal annealing temperature for the published primers was 55?C and the optimal annealing temperature for the designed primers was 65?C. There was a final elongation at 72?C for 5 minutes. A gel electrophoresis made with GreenGlo was run of the PCR product to determine amplification. All lanes show nonspecific binding when compared to the 1Kb Plus Generuler Ladder. Lane 2 represents non-specific binding because it contains a band at 365 bp, while the targeted region is 356 bp. The band for lane 2 showed more clearly with double the salt concentration (10μL PCR buffer and 35μL nuclease-free water). There is a band at 575 bp in lanes 3 and 5, which is a wild type DNA PCR product made with the normal salt concentration, while there are no bands in lanes 4 and 6, which is a PCR product made with double the salt concentration. Lanes 3 and 5 represent non-specific binding because the targeted region is 1183 bp. Lane 3 has two faint bands above the band at 575 bp, indicating more specific binding. Bands in all lanes ran past the 6x loading dye added to each PCR product, indicating the presence of primer dimers. Lane 2 shows a primer dimer at under 75 bp, while lanes 3 through 6 show primer