Identifying F2853S mutation of PKD in human S9 cells via site-specific primers in PCR and Gel Electrophoresis

By: Laura Kwasnik, Joy Burrell, Nick Rochte and Alex Reid

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disease that causes fluid-filled kidney cysts, ultimately causing renal failure. The F2853S mutation is a point mutation on the PKD-1 gene and affects Polycystin-1, which regulates cell growth and proliferation. (Braun, 2009). A diagnostic assay for this mutation was designed using allele-specific primers in PCR to screen for F2853S. The hypothesis was that the Yaku-designed primers would bind only to the targeted region of the PKD-1 gene because two mismatches between the primers and non-target DNA increase specificity. Site-directed mutagenesis was used to produce mutated DNA from wild-type DNA from human S9 cells. Agarose gel electrophoresis was used to analyze the PCR product. Twenty-two PCR trials were conducted. The results suggest that the control primer was the only primer to successfully anneal to mutant or wild-type DNA, because a 500 b.p. band occurred most consistently at 48°C. No bands appeared at the predicted location of 922 base pairs. Success in detecting PKD with PCR could lead to earlier diagnosis of the disease. A sociological experiment was conducted to illustrate the difficulty of following a PKD diet. It was predicted that students would prefer foods with higher sodium content than the recommended value of 1500 mg per day, and this was supported by a p-value of 0.00005216 (Figure 6). Blood pressure was tracked for a two-week duration of the diet to see if the diet led to a decrease in blood pressure but there was not a significant decrease because the p-value was 0.34910 (Figure 6).

Figures

Two Replications of identical PCR cocktail with annealing temperature of 48˚C with Lambda virus control. The three lanes label “L” all show an Invitrogen 1 Kb Plus DNA ladder used to measure the length in base pairs. Lane 1 contains wildtype DNA with wildtype primers, Lane 2 contains wildtype DNA with mutant primers, Lane 3 contains mutant DNA, F2853S mutation of the PKD-1 gene, with wildtype primers, Lane 4 contains mutant DNA with mutant primers. Lane 5 shows wildtype DNA with wildtype primers. Lane 6 contains wildtype DNA with mutant primers, Lane 7 contains mutant DNA with wildtype primers. Lane 8 contains mutant DNA with mutant primers. Lanes 9 and 10 contain two identical PCR cocktails of lambda virus. The DNA was run for 30 cycles. All of the products were exposed to the same PCR cocktail and same conditions. The denaturation phase was run for 45 seconds at 94°C. The annealing phase was run for 35 seconds; however each set of lanes had a different annealing temperature. Lanes 1-8 were run at an annealing temperature of 48°C; lanes 9 and 10 were run at an annealing temperature of 55°C, which is the optimum annealing temperature for lambda virus. The elongation phase was run for 45 seconds at 72°C. Then, the PCR products were placed into a 1% agarose gel with LB buffer and stained with ethidium bromide at run at 300V for about 10 minutes. Bright bands are shown at 500 b.p. at 48 °C in lanes 3, 4, 7, and 8 and faint bands are shown in lanes 5 and 6; these bands were from the control forward primer (CFP) which was designed to anneal at 500 b.p. Lanes 3, 4, 7, and 8 show streaks. These streaks could be from protein impurities in the DNA, or from impurities that occurred during mutagenesis. The annealing temperature of 48°C was found to be the most ideal for these PCR conditions. Lane 10 shows the expected 500 b.p. band for lambda virus. Lambda virus was used as a control; it contains the same PCR ingredients as the other lanes. This control was used to test to make sure none of the PCR ingredients were contaminated or old. Lane 9 is also lambda but does not show the expected 500 b.p. band because a hair was caught in the PCR tube when it was run through the PCR machine.

Discussion

Background Information

Polycystic kidney disease (PKD) is the most prevalent genetic kidney disease in the United States and affects about one in six hundred to one thousand people worldwide (Tufan et al., 2010, Garcia-Gonzalez et al., 2007). Mutations on the PKD-1 and PKD-2 genes cause mutations in the polycystin-1 and polycystin-2 proteins, which function in the membrane of the cilia of the kidney tubules (Braun, 2009). When these proteins are mutated, cell proliferation is not inhibited and fluid-filled cysts form on the kidneys which ultimately lead to kidney failure (Braun 2009). While PKD mainly affects the kidneys, cysts commonly develop on the liver and pancreas (Tufan et al., 2010). Other complications of PKD include hypertension, kidney stones, intracranial aneurysms, cardiac valve abnormalities, and hernias (Braun, 2009). There is currently no permanent therapy for PKD that can prevent the cysts from recurring (Takiar and Caplan, 2010). PKD genetic testing is limited, and currently ultrasound imaging is the most common way of diagnosing PKD (Garcia-Gonzalez et al., 2007). However, this often cannot be used to diagnose individuals under thirty years because the formation of cysts is age related (Garcia-Gonzalez et al., 2007). With further research and more complete understanding of the function of the polycystin-1 and polycystin-2 proteins, disease-specific therapies could be developed to treat or even prevent PKD.

The F2853S mutation is an acquired somatic mutation occurring on chromosome 16 at Exon 23 in which a single point mutation occurs, causing an amino acid substitution of phenylalanine for serine (Qian et al., 2002). The hypothesis was that the primers designed would bind to the targeted region of the PKD-1 gene where the F2853S single base pair substitution mutation occurs because of the two intentional mismatches in the forward primers for the mutant and wild-type DNA.

Experimental Predictions

It was predicted that the PCR would show a band at 922 b.p. for the correct pairing of mutant and wild type DNA with their associated primers because the forward and reverse primers were designed to anneal 922 b.p. apart. The Yaku method intentionally inserts two mismatches between a specific primer and the non-target DNA strand; therefore, it lessens the chance that the primers anneal to the non-target DNA (Yaku et al., 2008). A control primer was designed to amplify a band of 500 b.p. in every reaction which would serve as an internal positive control. If this positive control succeeded, it would show that the cocktail ingredients worked properly, that thermocycling conditions worked, that the gel electrophoresis was done properly, and that the reverse primer was functioning properly (since the control primer used the same reverse primer as the forward primers did).

Experimental Results

Site-directed mutagenesis was used to create mutant DNA template for the PCR assay because the F2853S mutation on chromosome 16 at Exon 23 was not able to be obtained from researchers. This mutant DNA was run with the PKDF primer and predicted to amplify a band of 922 b.p., and it was also run with the WTF primer as a negative control. The success of the site-directed mutagenesis procedure was not confirmed, so this was an experimental weakness.

A Lambda virus PCR cocktail was used as a positive control to ensure the viability of the PCR cocktail ingredients. In addition, the Lambda band of 500 b.p. was the approximate length of the control band. In the first Lambda virus gel (Figure 1), two bright bands appeared at the expected location. This is in error, because there should only be one band, and this could have been caused by an error in how the primers annealed to the DNA. However, this erroneous result did not affect the later PCR cocktails in which Lambda was used as a control, because different DNA template was used in the PKD PCR assay, and the correct bands were obtained. The bands were bright, indicating a large number of amplified DNA of a specific band length. Although different numbers of cycles were run, one lane was not noticeably brighter than another lane.

Genomic purification of S9 cells using the Quiagen Capture Column kit provided DNA template for the wild-type DNA and site-directed mutagenesis in the experimental assay. The 260/280 absorbance ratio, 1.284, indicates a low purity compared to the desired range of 1.4-1.9. This lower purity is due to proteins that were not completely extracted during the purification process. The yield of the purification process was high with a DNA concentration of 0.023 mg/mL, which allowed for many PCR trials to be run.

Six annealing temperatures were tested in the PCR assay. The positive control band of 500 b.p. was obtained in the gel with the annealing temperature of 52, 48, and 47 (Figure 2, Figure 3, Figure 4, Figure 5). The largest number of bands was consistently observed at 48 and 47 degrees. This showed that the reverse primer and control primer annealed properly, the cocktail ingredients were in the proper proportions, and that thermocycling conditions were optimized for these two primers. However, all of these factors were not discriminatory enough for the forward primers to anneal. A 922 b.p. band for the pairing of wild-type DNA with WTF primer or mutant DNA with PKDF primer was not observed. Therefore, the hypothesis that the Yaku method would provide greater discrimination was not confirmed, because no band was obtained using the Yaku primers. Overall, the assay did not function to detect the F2853S mutation.

Sociological Experiment

The DASH (Dietary Approaches to Stop Hypertension) diet, which is commonly used to lower blood pressure, was taken on by the researchers in order to experience a lifestyle effect of having PKD. Lowering blood pressure has been to shown to slow the progression of PKD (Schmid 1990). The DASH diet is based on a 2,000 calorie diet, and it suggests that people trying lower high blood pressure stay under 1500 mg of sodium a day (Heller 2004).

During the sociological experiment, each researcher followed the DASH diet for fourteen days. The blood pressure of each person was taken at the beginning and end of the diet, and they were compared to see if following the DASH diet lowered the blood pressure of the researcher. A p-value of 0.394 was obtained. The null hypothesis stated that there was no difference in the beginning and ending blood pressure, so it failed to be rejected. This indicates that the DASH diet was not effective in lowering the blood pressure of the researchers. However, since the diet was only followed for two weeks, better results may be obtained after a longer duration of following the diet. Also, since the researchers do not actually have kidney disease or hypertension, eating reduced sodium levels may not affect their blood pressure as profoundly.

The second part of the experiment included tracking each researcher’s sodium intake for a total of four days. The sodium levels for prepared meals served in the cafeteria were found on the Michigan State University Residential Dining nutrition website and the USDA MyPyramid Food Tracker. This aspect of the experiment assessed the difficulty of following a low-sodium diet while eating in the MSU dining halls. It was hypothesized that the actual amount of sodium consumed while following the diet would be less than the desired amount (or amount eaten under normal circumstances). A p-value of 0.0000522 was obtained, so the null hypothesis was rejected. This p-value shows that there was a significant difference between the the actual and desired amounts. However, there is some issue of bias in this calculation, since the researchers were aware of the guidelines and were able to plan their diet around the guidelines. Nevertheless, this large difference shows the drastic lifestyle change to reduce sodium levels to the 1500 mg recommended by the DASH diet to lower blood pressure and slow the progression of PKD.

Future Directions

One limitation of this experiment is that site-directed mutagenesis was not verified. If more time was available, restriction enzymes would be used to see if the mutation was actually inserted into the DNA strand. In addition, if the experimental primers amplified the expected 922 b.p. band in the future, restriction enzymes could be used to see if the correct band was being amplified.

After obtaining 500 b.p. control bands with 48 C and 47 C, troubleshooting was done to try to optimize PCR conditions for the WTF primer to anneal to the wild-type DNA template. Different DNA concentrations were used, but since the thermocycler was not set correctly, a 7-minute extension time was used and no bands were observed. Therefore, in the future, DNA concentrations would be altered in order to see if more DNA or less DNA would cause more distinct bands to appear for the control primer or a wild-type band. In addition, it was hypothesized that including more DNA in the cocktail would provide more DNA for the primers to anneal to.

The streaky lanes that resulted in several of the gels were present only in lanes that contained mutant DNA template. This is probably due to a contamination of the mutant DNA, which may have occurred during the site-directed mutagenesis process. There was no control for the cocktail ingredients used in mutagenesis. To troubleshoot this, we would run a PCR cocktail using lambda virus and the same ingredients in the cocktail as were used in the site-directed mutagenesis cocktails, and if the correct band was amplified it would show that the cocktail ingredients were viable.

On several of the gels there were no smaller bands at the bottom of the gel. The gel was completely empty of small bands (below 100 b.p.). This could have been due to lower primer concentrations than needed. In future experiments, concentrations of primers would be varied. If the correct DNA is amplified, this would show a brighter band. If the primers do not anneal to the DNA, more smaller bands would be evident at the bottom of the gel.