Assessing the Risk Factor of a Human Cell for HD by Using PCR to Identify the Number of CAG Expansions

 

 

 

By: Sean Benner, Ashley Grosso, Tluang Hniang, and Amy Jamieson

 

 

 

Abstract

 

Written by: A42663099

Revised by: A40407099

Finalized by: A40961460

            The purpose of our project is to calculate the number of CAG expansions that a genome contains to determine the risk factor for a cell with Huntington’s disease (HD). By conducting this study, advances can be made in the diagnosis of HD with the potential of discovering a cure. PCR will be used to replicate the CAG expansions, followed by gel electrophoresis to analyze the segment to see if a cell is at risk for HD. We hope that the primers designed will work in the PCR cocktail as evident by bands made by gel electrophoresis. We will be able to determine the number of base pairs based on a control band at 165bp from PCR product of the enamelin gene. Consequently, it will be possible to calculate the number of CAG and CCG repeats, thus determining if the cell is at risk for HD or not. We predict that the mutated cell will result in a band over 165bp, displaying a risk factor for the disease, while a normal cell will show a band less then 165bp, and therefore not be at risk for the disease. These values were determined by analyzing the lowest possible scenario for being borderline for HD, concluded to be 36 CAG and 6 CCG repeats (Watts and Koller, 2004). However, only primer dimer was achieved. A sociological experiment was conducted by utilizing a GAF test. It was ultimately found that when displaying symptoms of HD, an individual has a significantly lower level of functioning, as was originally predicted.

 

Discussion

Written by: A40407099

Revised by: A42663099

Finalized by: A42449618

 Research Summary and Hypothesis   

            Huntington’s disease is a fatal disease which is caused by a combination of CAG and CCG repeats found on gene IT15 at 4p16.3 on chromosome four. Both the number of CAG repeats and the number of CCG repeats are incorporated in the diagnosis of Huntington’s disease (Andrew et al, 1994). These repeats lead to the toxic effects of the protein huntingtin at various locations in the brain, particularly in the striatum and cerebral cortex. Huntingtin is important in the formation and development of the brain while in the womb as well as the survival of neurons in adults (Zuccato et al, 2001). Previous research has found an EcoP15I enzyme will cut at the CAG repeats to determine the number of CAG repeats. PCR and gel electrophoresis are another two methods for diagnosing a patient with Huntington’s. (Yen et al, 1999). The question we addressed was whether the primers we designed could be used successfully to determine a person’s risk factor for Huntington’s disease. We hypothesized that the Fprimer and Rprimer which we designed could be used to determine someone’s risk factor for Huntington’s disease by the process of PCR and gel electrophoresis. Gel electrophoresis from amplified DNA of Huntington’s disease was expected to display a band above 165bp using the HD forward and reverse primers while a band below 165bp was expected with the wild type DNA. To gain a better understanding of Huntington’s disease, a sociological study was conducted in addition to the laboratory experiment. Each researcher from the research team experienced the progressing stages of Huntington’s disease for a total of five days. The GAF test (Global Assessment of Functioning) was taken to evaluate the level of disability both psychologically and physically for each stage. It was hypothesized that the GAF test score will decrease for each individual as the symptoms of Huntington’s progress.

Original Predictions

            By interpretation of gel electrophoresis, the amplified DNA of infected Huntington’s disease and wild type DNA with the primers we designed were expected to determine the risk factor of a person’s having Huntington’s disease or not based on the number of base pairs shown. Because a normal person has fewer than 35 CAG repeats but greater than 6 CAG repeats, it was predicted that the normal gene sequence, those not at risk for developing Huntington’s disease, would show a band below 165bp. However, those at risk for Huntington’s disease were expected to show a band above 165b on the gel ladder because the correlating number of CAG repeats is greater than 40 (MacDonald et al, 1993). The forward and reverse primers for the enamelin gene were expected to create the band at 165bp as a positive control (Watts and Koller, 2004).

Primer Design

Using the HD genomic sequence, the forward and reverse primers were designed to attach before the CAG repeats and after the CCG repeats of the DNA sequence. In order for the primers to anneal properly, the temperature of the second step; annealing stage; of the PCR process was calculated by the melting point formula because theoretical melting point for primers is critical. If the temperature is too high, the primers won’t anneal properly to the DNA resulting in a bad PCR product. The same HD primers were used to attach to both the mutant and the wild type DNA because there is no difference between the two besides the number of CAG repeats where the mutant would have more than 40, while the wild type would have 35 or less. To make sure the PCR works and the primers anneal correctly, forward and reverse primers from the enamelin gene was designed as a positive control. For a negative control, the HD primers and enamelin primers were paired as forward and reverse primers in the PCR cocktails without DNA template to make sure there was no amplification.

Genomic Purification   

Douglas Luckie, a researcher at Lyman Briggs College of Michigan State University, provided the epithelial cells for wild type DNA samples. As the procedure of DNA purification found in the Generation Capture Column Kit from QIAGEN, the DNA was successfully extracted. The purpose of performing the DNA purification was to remove all unnecessary chemical that attached within the wild type DNA sample such as lipids, sodium, enzymes and proteins, and to obtain pure and desalted PCR amplicons (Manduzio et al, 2010). 

PCR and Gel Electrophoresis analysis

To determine the optimal PCR condition, PCR cocktails were run with multiple different annealing temperatures. Also, a 2% agarose gel was made for gel electrophoresis so that the small bands produced by PCR could be seen. In figure 2, the wild type DNA was run with multiple temperatures (45°C, 50°C and 56°C) to determine which temperature would result in the primers annealing properly and result in a band of the expected length. At every temperature, primer dimer was the result. The primers were annealing to each other instead of annealing with the wild type DNA (Huang et al, 2011).However, there is no primer dimer at lane 6 because something in the PCR cocktail was incorrect, possibly either the wrong primer was loaded or the amount of primers in the cocktail was not correct. Figure 3 shows a gel of negative controls run at 56°C and 60°C for Huntington’s and Enamelin primers. There were no bands shown as the primers designed from Huntington gene were not annealing to each other and the primers designed from enamelin gene were not annealing to each other. However, in the negative control shown in figure 4, primer dimer was evident when the HD and Enamelin primers were crossed in the cocktail. Our hypothesis was refuted by the lack of accuracy due to different outcomes of primer dimer in negative controls, the positive controls were not displayed as expected, and the wild-type DNA was not amplified with designed HD primers to result in a band below 165bp.

Sociological Experiment analysis

Along with working at the molecular level of Huntington’s disease, a sociological study was conducted. Figure 5 shows the results of an ANOVA (Analysis of Variance) statistical test ran using the GAF test scores taken after a normal day and the night of the second day during each of the three stages. The p-value calculated between the normal stage and each of the other three stages individually was found to be .08 for each test. Although that number shows no significance, when looking at the experiment as a whole the p-value was calculated to be .034, supporting our original prediction that there was a significant decline in functioning as the stages progressed. The third stage underwent another step of evaluation. The results from an ethogram taken during stage three of the sociological experiment are shown in Figure 6. The ethogram shows that the most common response from individuals was staring at the group member experiencing stage three. This was followed by avoiding eye contact, ignoring, and snickering at, while responses such as approaching and physically avoiding were hardly observed.

 

Future Direction

            Our experiment could be improved in many ways because when we performed our experiment, numerous amounts of error may occur from many different aspects. If there were six more weeks to work on the experiment and perform more PCR amplification, a perfect annealing temperature could be found. The HD primers could be redesigned so the band will be larger by placing the primers further upstream and downstream from the CAG repeats. Another way to improve the experiment is increasing or decreasing the amount of primers added into the PCR cocktails.  Furthermore, if we could have the mutant DNA available to us, we could have been able to amplify it in the PCR cocktail which might have resulted in bands with electrophoresis. Also, if we had access to upgraded PCR technology such as fluorescent multiplex PCR, the implication would have been more accurate results (Peciña et al, 2010). If for some reason the PCR and gel electrophoresis does not work, there are two alternatives that are plausible. The first of these would be to get the gene sequenced. There is a possibility that another lab would have the equipment to analyze our DNA samples and give us the specific sequence of our sample, allowing us to determine the number of repeats. This method has been employed by other scientists in the past (MacDonald et al, 1993). Another option would be the use of enzymes. Scientists have found an enzyme that cuts at the CAG repeats, resulting in gel bands representative of only the CAG repeats and nothing else (Yen et al, 1999). This could be used, if the EcoP15I enzyme is easily attainable, in the case that the PCR does not work (Möncke-Buchner et al, 2002). GAF test score might contain error due to self-scoring by the individual; it can be improved by accurate scoring by a professional.

 

 


Figure 4: Negative control gel containing different combinations of the Enamelin and Huntington’s primers. This gel depicts the results of a negative control ran containing different combinations of the two sets primers. In the 1st and 2nd lane there are the Enamelin reverse and Huntington’s reverse primers, in the 3rd and 4th lane there are the Enamelin reverse and Huntington’s forward primers, the 5thand 6th lane contain the Enamelin forward and Huntington’s forward primers, and lanes 7 and 8 contain the Enamelin forward and Huntinton’s reverse primers. The PCR was conducted using a Perkins-ElmerGeneAmp 2400 Thermo Cycler for 30 cycles. The denaturing was conducted at 95C for an initial 5 minutes and then 30 seconds for each denaturing cycle, the annealing temperature used was 57C for 30 seconds each annealing cycle, and the temperature for the extension portions was 72C for 30 seconds each extension cycle, and an additional extensions was conducted at 72C for 5 minutes. Annealing of the primers can be observed in all 8 lanes. The bands in lanes 1, 2, 5, and 8 are brighter, suggesting that more primers were annealing to each other in those lanes compared to lanes 3, 4, 6, and 7.