Genotypic Identification of Exon 41 of Human Wild-type LRRK2 in IB3-1 Cell DNA by PCR Amplification
By: Grace Barrett, Adam Panaretos, Julius Edwards, and Phalguni Kulkarni
Team Edgar Degas
LB 145
Monda y/Wednesday 7pm-9pm
Abstract:
The leucine-rich repeat kinase 2 (LRRK2) gene cod es for dardarin, an important protein located on exon 41 that helps to regulate dopamine levels in the human brain (Di Fonzo et al., 2005). When mutated, it is known to cause Parkinson’s disease. In order to provide more research towards the cause of Parkinso n’s Disease through human LRRK2 genetic amplification, we determined its genetic length using PCR and methods of DNA purification. Using published PCR met hods with published forward and reverse primers, we hypothesized that we would achieve successful amplification of exon 41 of the target LRRK2 gene via PCR, with the manipulation of concentrations and temperatures of each stage of PCR (denaturation, annealing, and elongation). Also, we hypothesized visual ization of the products acquired from PCR through gel electrophoresis will project the targeted DNA, approximately 349 base pairs because the exon 41 portion has been verified to be 349 base pairs long (Belin et al., 2006. In order to test these hypotheses, PCR was implemented and followed by gel electrophoresis to amplify and examine the target region of the LRRK2 gene. Before endeavoring to amplify the LRRK2 gene, the RZ1 gene of the lambda virus was amplified at approximately 400-500 bp to initially be used as a positive control for our experiment. However, the base pair length was deemed inconclusive due to inadequate ladder separation, which led to an incorrect base pair calculation when using the semi-log plot of the 1 Kb Plus DNA ladder. After experimenting with amplification of the RZ1 gene, cloning of exon 41 of the LRRK2 gene began. After analysis of our products, we concluded it showed non-successful DNA amplification of the exon 41 portion of the LRRK2 gene. The gels from experimentation showed no sign of amplified DNA. The goal of replicating exon 41 of the LRRK2 gene was unsuccessful, but with additional time and experimentation, amplification could be achieved to fulfill the overall goal - to provide more research into the cause of Parkinson’s disease - of the experiment.
Discussion:
Experimental Summary
Parkinson’s disease is a debilitating disease that occurs due to a base pair replacement in the exon 41 region of the LRRK2 gene. Ultimately, this leads to a loss of muscle control because the LRRK2 gene can not efficiently code for dardarin, a signaling protein primarily used in cell communication (Belin et al., 2006). The inadequate functioning of dardarin leads to nigra cell loss, which then leads to lower dopamine levels causing fewer neurons to be active (Chinta and Andersen, 2005). The L-Dopa drug is most commonly prescribed along with surgically creating lesions in certain brain regions (Chinta and Andersen, 2005). There is no absolute cure for Parkinson’s disease. In order to further research and understand this disease, our experiment employed PCR to amplify the exon 41 region of the target gene LRRK2. The purpose of this experiment was to use published protocols to successfully amplify the target gene. We hypothesized that manipulation of temperatures and concentrations of PCR would allow successful amplification of the LRRK2 gene, thus yielding bands at 329 base pairs using gel electrophoresis (Belin et al, 2006).
Original Predictions
Using an original PCR protocol from published research and designing our own specific forward and reverse primers, we predicted that amplification of the LRRK2 gene would be seen at 329 base pairs (Belin et al, 2006). Amplification of the LRRK2 gene would be visualized using gel electrophoresis. Also, we predicted that running a multitude of trails attempting amplification of the Rz of lambda would ultimately be successful due to multiple manipulations of concentration, amounts and temperatures of PCR (Gouvea et al, 1990).
Results and Key Findings
PCR was used to amplify the Rz gene of bacteriophage Lambda (Figure 1a). Amplification of the Rz gene served as positive control because it verified that our combination of an effective PCR cocktail and temperatures was successful in obtaining bands. Hence, this PCR protocol could be replicated for successful amplification of the LRRK2 gene of Parkinson’s Disease. The amplification of the Rz gene was a success because we obtained distinct bands which can been seen approximately around 500 base pairs (Figure 1a). The exact length of the Rz gene is 459 basepairs (Taylor et al, 1983). A molecular size vs. molecular migration semi-log plot was utilized and calculations were made using trendline equation (Figure 1b). The indistinctive separation of the ladder caused the equation of the semi-log plot to yield an insignificant and futile result, of 3.39x10-4 base pairs, upon calculation using the trendline. Therefore, we could not conclude this data as significant because the 1 Kb Plus Ladder was not distinctly separated. This forces us to deem our positive control unsuccessful. We believe that a more clear, distinct separation of the ladder would have yielded more reliable results. This would lead to extraction of more sufficient information from the more distinct base pair markers for a more reliable trendline equation. Also, a more distinct ladder would have allowed us to be able to defend the amount of base pairs the Rz gene travelled due to more sufficient information obtained from more distinct base pair markers. The ladder could have been improved using a higher concentration of agarose along with experimenting with different concentrations of 1 Kb Plus ladder in junction with the loading dye. A dilution method of the ladder could have been utilized in order to get clearer, more precise base pair markers (Chen et al, 2004).
The same PCR cocktail used for the positive control was used for the negative control void of any DNA template (Figure 2). The remaining wells, 2 through 5, contained the same PCR cocktail as our positive control. Slight smearing present in these lanes indicates that the primers annealed to themselves. Unsuccessful amplification of the Rz gene, in this case, could be caused by inaccurate denaturing, annealing, and elongation temperatures and time. The negative control is depicted in lane 6 of Figure 2. The negative control was successful because no bands appeared - the absence of DNA is responsible for this. As expected, the primers were not able to replicate anything.
In order to attempt amplification of the LRRK2 gene, DNA from human IB-3 cells was purified and then analyzed using the Epoch Spectrophotometer. An A260/280 value represents the amount of DNA and protein present in the cellular solution. The absorption spectrum of 260 nm indicates the presence of DNA and 280 nm indicates the presence of protein. A range of 1.8nm to 2.0nm indicates sufficient purification of the DNA. Our first genomic purification yielded an average 260/280 value of 2.026 (Table 1). Our second genomic purification was more successful and yielded an average 260/280 value of 1.749 (Table 2). However, we were not successful in amplification of the LRRK2 gene (Figure 4). One weakness of our experimental design was the fragility of the gel electrophoresis; it is essential to be careful not to tear the slots when exporting our PCR and our control ladder into the slots for illumination (Belin et al, 2006). This human error was corrected by honing our pipetting skills in order to refrain from disturbing the gel. Another weakness was miscalculation of primer heat (Taymans et al, 2006). This was remedied by researching other possible primer temperatures and testing them with several PCR cocktails to find the temperatures that would yield the clearest bands in our gels.
Successful amplification of the Rz gene indicated that our hypothesis was not refuted. Given that the semi-log plot did not provide the evidence that was needed to deem amplification of the Rz Lambda gene as a positive control, it should still be noted that clear bands can be seen (Figure 1a). The only problem that arose with our positive control was inadequate separation of 1 Kb Plus base pair markers. This can be solved by acquiring a better ladder through more time, more trials, and manipulations of concentration. This would then lead to a more accurate trendline, which would allow us to calculate the correct base pair length the Rz gene travelled.
Future Directions
The main limitation of our research was time. We encountered amplification of the Rz gene after approximately 15 attempts. We only attempted amplification of the target LRRK2 gene twice. More time would allow us to further investigate amplification and experiment with different concentrations of PCR ingredients, along with different temperatures. Trails would be run until successful amplification of the LRRK2 gene which would be evident in clear, precise bands. Amplification of the LRRK2 gene is possible and is relatable to past research such as that of Köhler et al, Cheung and Nelson, and Belin et al. Belin and colleagues not only cloned and studied the mutation G2019S within the LRRK2 gene but also the wild-type LRRK2 as well, which is substantial to our research in cloning the wild-type LRRK2 gene (Belin et al, 2006). A future possibility for the continuation of our research could be an experiment aiming to compare the mutated gene to the wild-type gene to contribute observations and findings that may lead to the discovery of a treatment or cure for Parkinson’s disease. Additionally, our research could help facilitate a greater understanding of Parkinson’s disease while adding to the existing research for the scientific community to use.
Figure 1a: Targeted amplification and analysis of bacteriophage lambda Rz gene using PCR and Gel Electrophoresis. The target DNA sequence, the Rz gene, consisted of 459 bp and was amplified using PCR in the Bio Rad Thermo-cycler. The PCR cocktail contained 38µl of distilled water, 7µl of 10X PCR buffer, 1µl Taq polymerase, 1µl dNTPs, 1µl of lambda DNA template, 1µl of 1Rz1F (forward primer), and 1µl of 1Rz1R (reverse primer). This mixture was run through the thermocycler for an initial period of 5 minutes at 95°C, followed by 30 cycles of 30 seconds of denaturation at 95°C, 30 seconds of annealing with a gradient of 50.0°C-57.4°C, and 1 minute of elongation at 72°C. The different temperatures resulting from the gradient are noted above each well. A 0.8% agarose gel was made by diluting 10µl 10X TBE (Tris/Borate/ EDTA) buffer with 90µl distilled water in order to make 1X TBE Buffer. 1µl GloGreen gel stain and 0.4g agarose powder were added to this dilution. The gel contained 3µl 6x purple loading dye and 7µl PCR cocktail in wells labeled 2-8, and 5µl of 1Kb Plus ladder along with 1µl 6x purple loading dye in well 1. The products in the gel were visualized using the Bio-Rad Gel Doc EZ.
Figure 1b: Semi-Log plot used to calculate the distance traveled by target Rz gene of bacteriophage lambda. A target sequence of the Rz gene of bacteriophage lambda was amplified using PCR. This semi-log plot, created using Microsoft Excel, analyzes the migration distance vs. molecular size of the 1Kb Plus ladder (Figure 1a). The x-values represent the distance each band traveled away from each well. The distance was measured in centimeters, starting at the bottom of the well and ending at the bottom of the band. The y-values represent the molecular size of each band and is measured in base pairs. A trend line was created in order to further analyze the results. The equation obtained from the trend line (y = 10098x-4.88) was used to calculate the molecular size of the fragments in lanes 2-8. The distance that each band migrated from its well was used as the input value for x, which yielded a y-value revealing the base pair length of the target gene. The base pair length calculated according to the equation was inconclusive. An R² value of 1 represents the perfect fit of the trendline; the R² value of 0.861 shown in the figure represents the best fit of our results.
