An Unsuccessful PCR Detection of the CGG Trinucleotide Repeats on the FMR1 Gene Using Various Homo Sapien Cells

By: Kaitlyn Beels, Kelsey Licht, Alyssa Seel, Justin Zaleski

Abstract

The CGG trinucleotide repeat on the Fragile X mental retardation gene (FMR1) causes the genetic sex-linked disease, Fragile X (Dodds et al, 2009). Individuals affected by the disease have over 200 CGG repeats on the FMR1 gene (Hawkins et al, 2010). An experiment was designed with the purpose of differentiating between DNA from a Fragile X patient and a normal individual using polymerase chain reaction (PCR) and gel electrophoresis. It was hypothesized that the amplified products of the mutant DNA would have a longer sequence than those of the wild type DNA and therefore, when run through agarose gel would appear as a band closer to the well. It was predicted that this would be due to the additional CGG repeats within the mutated DNA (Filipovic-Sadic et al, 2010). Primers were designed to anneal to the FMR1 gene of both wild-type and mutant DNA. The products were run through a 1% agarose gel. Results showed no desired amplified PCR products. Future research could include more experimentation with annealing temperatures, hot start PCR, or redesign of primers. If future research led to desired amplification, this method could be used to diagnose patients with Fragile X. A sociological experiment was run where group members performed activities that drew negative attention to themselves to gain a better understanding of what Fragile X patients deal with on a daily basis. Results showed a positive correlation between the amount of negative attention received and the level of discomfort experienced, with an R² value of 0.7573.

Discussion

Background and Hypothesis

Fragile X is caused by a trinucleotide CGG repeat on the FMR1 gene. When an individual has over 200 CGG repeats, they have full mutations of Fragile X. Those with pre-mutations have between 50-200 repeats and normal individuals generally have 6-50 repeats (Garber et al, 2008). Symptoms of Fragile X include long or large facial features. Other symptoms include impaired learning and motor skills along with behavioral problems. These symptoms can bring negative, unwanted attention to these individuals (Aziz et al, 2003).

In this experiment, PCR was used to amplify a target sequence of DNA from the FMR1 gene to distinguish between DNA from a Fragile X patient and an individual without Fragile X (Wu et al, 1989). Primers 1 and 2 were designed to anneal to both mutant and wild-type DNA. It was hypothesized that the primers would amplify 220 base pairs with wild-type DNA and at least 880 base pairs with mutant DNA and therefore, the mutant DNA would show up closer to the wells when run through a 1% agarose gel. It was predicted that the mutant strands would be longer due to the additional CGG repeats (Filipovic-Sadic et al, 2010). Primers 3, 4, and 5 were designed to amplify 430 and 880 base pairs from chromosome 8 (representing 50 and 200 trinucleotide repeats) to differentiate between individuals without Fragile X, with pre-mutations, and with full mutations. This served as a positive control. Mixtures were created to run in PCR, and then the amplified products were run through a 1% agarose gel. A negative control was also used in the experiment. Reaction mixtures were created without any template DNA. This was run through PCR and then through the 1% agarose gel. With desired amplification of the DNA target sequence, this experiment could be used to diagnose a person with Fragile X Syndrome because the additional CGG repeats would be observed (Filipovic-Sadic et al, 2010).

A sociological experiment was designed to allow group members to put themselves in the shoes of a Fragile X patient. All group members did something to bring unwanted attention upon themselves around campus. Members then rated how uncomfortable they felt on a scale of 1 to 5 with 5 being the most uncomfortable. This was to help understand the negative attention Fragile X patients experience, leading to their discomfort. It was hypothesized that members would feel more uncomfortable with increased negative attention from being outside of their comfort zones.

Experimental Analysis and Troubleshooting

In the first trial after 30 cycles with an annealing temperature of 54°C, no bands were seen in the lane containing primers 1 and 2 where there should have been a band at 220 base pairs. It is possible that gel electrophoresis was run for too long, causing the DNA to run off the gel. To avoid this in the next trial, Orange G loading dye was used in place of Bromophenol Blue to allow for better monitoring of the progress of DNA on the gel. All other conditions were kept the same for this mixture. In the lane containing primers 3, 4, and 5 where there should have been bands at 430 and 880 base pairs, there was non-specific binding. For additional troubleshooting, it was decided to increase the annealing temperature for this mixture containing primers 3, 4, and 5 from 54°C to 57°C (Wittwer et al, 1993). Then, one mixture with primers 1 and 2 was created with the same components as the previous trial. Another mixture with primers 1 and 2 was created, but with 1 µl Dimethyl Sulfoxide (DMSO). Similarly, one mixture with primers 3, 4, and 5 was created without DMSO as before, and another was created with the DMSO. DMSO is a chemical that can help improve amplification of DNA containing high C-G content by helping to separate the three hydrogen bonds between each pairing of C-G nucleotides. DNA templates containing C-G rich regions often give weak signals without additives such as DMSO (Sahdev et al, 2007).

The second trial resulted in no bands. The lack of success in this trial could have resulted from increasing the annealing temperature by 3°C when the better lab technique would be to increase the temperature by 1°C or 2°C. Also, the lack of bands could have resulted from a low concentration of purified DNA in the sample. The first sample of wild-type DNA contained 0.004 mg/mL DNA. To improve this purity, more wild-type DNA was extracted from hair follicles using a Chelex solution. This sample contained 0.009 mg/mL of DNA. This was used in the next trial because it was more pure. In addition, the annealing temperature was decreased back to 54°C for all the samples. This was the only change made for the next trial.

The third trial resulted in no bands. After researching for solutions, a two-stage thermocycling procedure was found to successfully amplify the CGG repeat in Fragile X (Todorov et al, 2010). In hopes that this procedure could yield successful results, this was used in the following trial and the annealing temperature was kept at 54°C. Mixtures with and without DMSO were still included because it was unknown which condition would give the best results.

The fourth trial included both wild-type and mutant DNA. The two-stage thermocycling was performed and primer dimer occured for the mutant DNA and the wild-type DNA with primers 1 and 2. The lane containing wild-type DNA with primers 3, 4, and 5 without DMSO had faint bands at the desired target sequence of 430 and 880 base pairs, but had non-specific binding. Primer dimer and non-specific binding can occur due to an annealing temperature that is too low (Mitra and Church, 1999). For the next trial, the annealing temperature was increased to 56°C for all of the samples while continuing to use the two-stage thermocycling because it produced more bands than the traditional single-stage thermocycling procedure.

The fifth trial resulted in no bands. It is possible that the annealing temperature was too high because that can prevent the primers from binding to the template DNA (Cao et at, 2004). With time for only one more trial, the annealing temperature was dropped to 51°C in hopes of seeing some bands. In addition, the Taq polymerase concentration was doubled in those mixtures containing DMSO because DMSO can denature Taq (Sun et al, 1993).

The sixth trial showed no bands. It is possible that the annealing temperature was still too high, preventing binding from occurring. The annealing or denaturation temperatures could have also been too low, preventing the DNA from unwinding, and the primers from annealing. These variables could be changed during future research.

Ultimate Findings

No successful bands were seen with primers 1 and 2 for both wild-type and mutant DNA. Bands were found with primers 3, 4, and 5 at 430 and 880 base pairs with the wild-type DNA from chromosome 8, however they were faint. This means that although our positive control was successfully amplified, our data is inconclusive because no bands formed with primers 1 and 2. We were unable to distinguish between the DNA of a Fragile X patient and a normal individual. This does not mean PCR is an ineffective method for diagnosing Fragile X Syndrome, but rather that more trials would need to be performed.

In the sociological experiment, the average level of discomfort for each trial was plotted on a graph against the number of times negative attention was directed at the participant. The results showed a positive correlation, given by an R² value of 0.7573, between the amount of negative attention received and the level of discomfort experienced. This means that the more negative attention that is received, the more uncomfortable an individual will likely feel. As Fragile X patients bring attention upon themselves as a result of their behavior or abnormal symptoms, they experience extreme discomfort because they tend to dislike being the center of attention.

Future Research

If more time was available to continue the research, different annealing temperatures could be used because the optimal temperature was not yet discovered. In addition, the primers could be diluted more because at high concentrations, primers can bind to each other, forming primer dimers (Mehra and Hu, 2005). A “hot start” PCR procedure could also be used. This procedure involves withholding the Taq polymerase until the reaction temperature has reached 80°C. It has been used to improve amplification of C-G rich regions of DNA and it reduces off-target amplification (Ashrafi and Paul, 2009). New primers could be designed in a location where it is least likely to produce non-specific binding (Shigemori et al, 2005). This would be especially useful for primers 1 and 2 because no trials showed successful bands using these primers. Therefore, it is possible that something was wrong with their design.

For further research in the sociological experiment, a Fragile X patient could be shadowed in order to see firsthand how they are treated and how it makes them feel. We could also look at how caretakers or parents are affected by the negative attention that is brought upon the patient. In addition, a mother of three children with Fragile X offered further research ideas including an activity with three participants. Participant 1 is given the role of a Fragile X patient but is not told this is their role. Participant 2 plays the environment and pokes, tickles, blows on, and tries to annoy Participant 1. Participant 3 plays a person of authority and consistently asks any type of question they want. This continues for a couple minutes until Participant 1 is distressed. Then Participant 1 can tell how they felt during the activity. This is often how a Fragile X patient feels on a day-to-day basis (Personal Communication, wishes to have name remain anonymous).

FIGURE 1.

1/M L2 L3 L4 L5 L6 L7 L8

Figure 1. Amplification of mutant and wild-type DNA, trial 4. Lane 1 shows a 1 KB plus molecular weight ladder. Lane 2 and 3 contain mutant DNA with primers 1 and 2. Lanes 4 and 5 contain wild-type DNA with primers 1 and 2. Lanes 6 and 7 contain wild-type DNA and primers 3, 4, and 5. Lane 8 serves as a negative control with a PCR mixture with primers 1 and 2 and no DNA. DMSO added to the PCR mixtures that were run through lanes 2, 4, and 6. A two-stage cycling procedure was used to analyze the PCR products. For the first 10 cycles, the reactions went through denaturation for 35 seconds at 97°C, annealing for 35 seconds at 54°C, and extension for 4 minutes at 68°C. For the following 25 cycles, they again went through denaturation for 35 seconds at 97°C, annealing for 35 seconds at 54°C, and then they went through extension at 68°C for 6 minutes. The products were then run through a 1% agarose gel using TBE buffer. Primer dimer was seen in lanes 2, 3, and 5. Faint bands were seen at 880bp and 430bp in lane 7, which is what was intended, along with non-specific binding which was undesired.