Gel electrophoresis shows PCR a valid method of amplifying

FGFR3 gene in IB3 cells

By: A44540941, A46720223, A47772652

Abstract

The FGFR3 gene, or fibroblast growth factor receptor 3, codes for a protein which regulates cell growth by preventing ossification in the bones (L’Hôte and Knowles, 2005). A Gly380Arg mutation of this gene creates an overactive FGFR3, which stops ossification prematurely (Horton et al, 2002).  This mutation is responsible for over 97% of all achondroplasia, or dwarfism cases (Vajo et al, 2000). In this study, the wild-type FGFR3 gene was amplified using polymerase chain reaction (PCR) methods, which are commonly used to identify and diagnose a patient with achondroplasia. DNA was purified and extracted from Homo sapiens IB3 cells using a QIAGEN Generation Capture Column Kit and amplified using PCR with previously designed primers for the wild-type FGFR3 gene (Ikegawa et al, 1995). The products were analyzed using agarose gel electrophoresis. Amplification in the gel was detected by UV-visible bands that were measured by comparing them to a ladder of known base pair lengths. The visible band showed amplification of about 200 base pairs. To verify the quality of the materials, the 16s rDNA gene of Escherichia coli has been used as a positive control. We hypothesized that if we controlled the reaction cocktail and annealing temperatures, the published primers for the FGFR3 gene would successfully amplify the gene through PCR.  Successful amplification is significant in enabling further studies regarding treatment for achondroplasia.

Discussion

Experiment Summary

The FGFR3 gene codes for the fibroblast growth factor receptor 3 protein, which regulates the growth of various cells (Deng et al, 1996). It is especially important in limiting the growth of bone cells by preventing ossification (L’Hôte and Knowles, 2005). The gene is 2,520 nucleotides long, and the protein is 840 amino acids long (Vajo et al, 2000). In over 97% of cases of achondroplasia, the most common form of dwarfism, the patient has a Gly380Arg mutation in the FGFR3 gene (Vajo et al, 2000). Studies suggest that this mutation works by stabilizing FGFR3 dimers or recycling activated receptors, thus increasing both the intensity of the signal and the lifetime of the gene (Horton and Lunstrum, 2002). PCR has been used as a diagnostic method to amplify the mutated FGFR3 gene in achondroplasia patients (Patil et al, 2009). Thus, we hypothesized by regulating the PCR cocktail, annealing temperatures and using the published primers, then we would successfully amplify the wild-type FGFR3 gene using PCR methods (Ikegawa et al, 1995).

Original Predictions   

For the positive control, the 16s rDNA gene of E. coli was amplified using PCR. If successful, it was expected that the agarose gel of our product would produce one band.  We hypothesized that if the 5’ end of the 11F primer annealed to the 11^th nucleotide  and the 5’end of the 529R primer annealed to the 529^th nucleotide, then it would result in a PCR product 518 base pairs in length (Igert et al, 2015). This would help verify that the PCR materials and equipment, such as thermocyclers and agarose gel kits, function normally. By amplifying the DNA from IB3 cells, it was expected that successful PCR for the wild-type Homo sapiens FGFR3 gene in IB3 cells would result in a product that would produce one band. We predicted that an agarose gel would reveal a fragment 200 base pairs long (Ikegawa et al, 1995).  

Results and Ultimate Findings

The results show multiple attempts to produce the correct PCR products for the 16s rDNA gene of E. coli and the wild-type FGFR3 gene. Multiple trials were run for the E. coli positive control. The early trials of gel electrophoresis for the 16s rDNA gene resulted in mostly primer dimer with no visible bands, but a large amount of DNA remaining in the wells of the agarose gel. To troubleshoot the problems we adjusted the annealing temperatures and the loading dye to DNA ratio for almost every trial. Theses changes produced the most visible bands for the 16s rRNA gene. This resulted in a band in well 2 that was 608 base pairs and a band in well 3 that was 635 base pairs, both of which were larger than our predicted band length of 530 base pairs (Figure 2A). These results contradict our prediction for the 16s rDNA gene because the bands were the wrong product size, which could have been a result of unspecific binding.

In our first attempt at amplifying the FGFR3 gene, the gel electrophoresis revealed a faint band with a length of 879 base pairs, with smearing and primer dimer (Figure 3A). The annealing temperatures were adjusted in order to produce a more distinct band, which ultimately resulted in no band with increased smearing and primer dimer in each well. In an attempt to get rid of the smearing we increased the amount of DNA in the cocktail and lowered the PCR cycles. The result of the adjustment did not decrease the smearing. Although the issue with smearing was never resolved, the final gel electrophoresis for FGFR3 revealed a 200 base pair band, which matched our predictions (Figure 4A). The correct band length could be due to the change in the amount of time the primer annealed during the PCR cycle. In the initial PCR trials for the FGFR3 gene, the annealing cycles only lasted for one minute, whereas in the final trial the annealing cycles lasted one minute thirty seconds. Also, the annealing temperatures were lowered for the last trial of the FGFR3 gene when compared to the first trial. The successful amplification of this gene supports our hypothesis because when we controlled the PCR cocktail and annealing temperatures, and used the published primers, the FGFR3 gene was correctly amplified.

Future Directions

Although the FGFR3 gene was successfully amplified, there are still several changes that could further improve the experiment. The most prominent problems were primer dimer in the gel, no visible bands, or bands that were incorrect in size. For our positive control the distinct bands were the wrong size. When bands are not the predicted lengths, it would imply that nonspecific binding occurred between the primers and the DNA sample, creating undesired products. If the annealing temperature is too low due to calculation or machine error, or if the concentration of magnesium ions is too high in our buffers, then nonspecific binding may occur (McPherson and Møller, 2000).  Raising the annealing temperature or adjusting the ionic concentrations in the PCR cocktail are possible ways to resolve the problem (Howe, 2007).

If no bands are present in the agarose gel electrophoresis, then we could obtain new DNA samples to purify to try and eliminate error due to degradation, or adjust the concentration of primers used (Canene-Adams, 2013). For primer dimer, the primers attach to themselves instead of the DNA template during PCR. This decreases the amount of primer in the reaction cocktail and  produces non-specific binding (Simantini et al, 1999). To reduce the amount of primer dimer, the addition of a sequence at nucleotides at the 5’end of the primers can suppress the formation of primer dimer (Simantini et al, 1999). Also, adjusting the annealing temperature and adjusting the concentration of primers (McPherson and Møller, 2000). If the concentration of primers is too low, there may not be enough primer to synthesize the desired amount of product, and if the concentration is too high then the primers may begin attaching to each other rather than the target DNA.

If research were to continue, in future experiments we would explore the use of alternative PCR methods such as hot-start PCR. Hot-start PCR withholds Taq from the mixture until the thermocycler exceeds the optimal annealing temperature, helping prevent problems such as nonspecific binding and smearing (Howe, 2007). The use of another PCR technique could be used to achieve the desired PCR products.

Figures

Figure 4: Amplification of FGFR3 gene in IB3 cells using previously published primers. (A) Agarose gel electrophoresis of PCR products. The PCR cocktail contained 36µL of water, 5µL of 10X PCR buffer, 1µL of dNTPs, 3µL of forward primer, 3µL of reverse primer, 1µL of purified IB3 genome, and 1µL of Taq polymerase. The cocktail underwent initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing over a gradient from 50°C-57°C for 1 minute 30 seconds, and extension at 72°C for 1 minute 30 seconds. After the 35 cycles were complete, the cocktail underwent another extension at 72°C for 5 minutes, and then held at 12°C until the agarose gel was ready. A 1% agarose gel was made by combining 1.2 grams of powdered agarose with 120mL of 1X LB (lithium boric) buffer and heating until the agarose dissolved. The mixture was cooled briefly and 3µL of 10mg/mL GloGreen dye was added. Immediately after adding the GloGreen dye, the mixture was swirled, poured into a gel tray, and allowed to set. Once the gel had solidified and immersed in 1X LB buffer, 1µL of 6X loading dye and 4µL of 100bp ladder were mixed and pipetted into wells 1 and 10, and 1µL of loading dye and 3µL of PCR product were pipetted into wells 2-9. The PCR products pipetted into wells 2-9 differ by the temperature they underwent annealing: the product from well 2 underwent annealing at 57.0°C, the product from well 3 underwent annealing at 56.5°C, etc. Bands around 200 base pairs are boxed in red and can be seen in all lanes with varying degrees of distinctness. All lanes contained smearing and primer dimer. (B) A semi-log plot based on a 100bp ladder shows the correlation between molecular size (y-axis; bp) of the DNA fragments vs. the distance migrated through the well (x-axis; cm). Using the trendline, the bands were shown to be 200bp long.