Using PCR to Successfully Identify the G1138A Point Mutation on the FGFR3 Gene in Achondroplasia Human Cells

Team Dead Matadors:

Nick Hadorn

Shubhi Kalwani

Rachael Hume

Melanie Hardin

Abstract

The FGFR3 gene on chromosome 4 controls the ossification of long bones and when a single point mutation at nucleotide 1138 changes guanine to adenine, it alters the protein sequence by coding for arginine rather than glycine. The result is Achondroplasia, which is the most common form of dwarfism (Etlik et al., 2008). We seek to produce an experiment that will correctly identify the G to A point mutation that causes Achondroplasia. Two forward primers were designed using the Yaku-Bonczyk method, one annealing to the wild DNA sequence and one to mutated DNA. A traditional reverse primer was also designed to work with both sequences. When wild type primers anneal to wild type DNA and when mutant primers anneal to mutant DNA, a band will result during gel electrophoresis at specific target zone due to the specific primer design (Yaku et al., 2008). The forward primers bind at base pair 1125 and the reverse binds at base pair 1775, creating a 653 base pair marker (Lanning and Brown, 1997). Following the Qiagen handbook protocol, 0.08 μg of human IB3 DNA was extracted using the Generation Capture Column Kit. Gel electrophoresis was used to test the DNA, following PCR amplification. A double blind experiment was successfully performed to test the validity of our primers as a diagnostic assay. Additionally, a social simulation was performed to better the understanding of the obstacles Achondroplasiacs face in daily life. Yaku primers, a double blind experiment, and social simulation were used to advance the current diagnostic assay research on Achondroplasia.

Figure 5. Double blind experiment with mutant primers and unknown DNA samples to predict presence of the G1138A mutation. Six PCR cocktails were prepared with mutant forward primer and reverse primer and no DNA. Without knowledge of which DNA was wild type DNA or mutant DNA, 1.25 ng of wild type DNA or 1.0 ng of mutant DNA were added to each PCR cocktail and it was run through the thermocycler with an annealing temperature of 51°C and analyzed using a 1% agarose gel run at 121V. The first and last well contained 5 μg of 1 Kb Plus DNA ladder. Each lane was loaded with 10 μL of PCR product containing 0.25 ng of DNA and mixed with 2 μL of bromophenol blue 6x loading dye. Lane 2, lane 3, and lane 6 presented bands at 653 base pairs signifying that mutant DNA was added to the PCR cocktail. Lane 1, lane 5, and lane 6 did not present defined bands at 653 base pairs but there was non-specific binding throughout the lane signifying that wild type DNA was used.

Discussion

Achondroplasia is the most common form of dwarfism (Etlik et al., 2008). It is caused by a single point mutation on the FGFR3 gene, which leads to a substitution of adenine rather than guanine at base pair 1138. The allele that causes this disease is autosomal dominant, and when a child receives two mutant alleles, most die shortly after birth (Lanning and Brown, 1997). The mutation prohibits chondrocytes from ossifying empty space, which impedes the long bones from growing, resulting in shortened limbs (He et al., 2010). By using various methods such as DNA extraction, PCR, and gel electrophoresis, an approach to effectively identify mutated DNA can be developed and utilized. In order to establish that this research assay can be used in diagnostic analysis, a double blind experiment was performed. To advance the current diagnostic assay on Achondroplasia, a social simulation experiment was used to investigate how people with this mutation are affected (Wu et al., 1989).

Primer Design

Specifically designed Yaku-Bonczyk primers were made to anneal to both the wild type and mutant type DNA and amplify the 653 base pair product. This method of primer design was selected because the mismatches at the first and third nucleotide from the 3’ end improve specificity when the primers are annealing (Yaku et al., 2008) (Figure 1). Before ordering the primers from IDT, they were verified using the NCBI primer BLAST program. When combined with the reverse primer designed to anneal to both DNA types, the appropriate base pairs were amplified and bands appeared in the agarose gel. There was non-specific binding present in some trials, which could have been caused by the reverse primer. This primer had several cytosine and thymine repeats allowing it to anneal to various locations on the gene and present base pair bands at incorrect markers on the gel (Etlik et al., 2008) (Figure 3) . This non-specific binding could be eliminated with a more carefully designed reverse primer with fewer base repetitions (Wittwer et al., 1993). This would decrease the probability of the primer annealing to the wrong base pairs. The forward mutant primer correctly annealed to the sequence, but occasionally misprimed. This could be due to the two base pair mismatch on the primer not being adequate to prevent extension, resulting in unexpected amplified product (Etlik et al,, 2008) The ultimate findings were conclusive with the original predictions, indicating this method of primer design was appropriate for the point mutation.

PCR

PCR was used to amplify a target zone on exon 10 of the fourth chromosome of the human genome. This amplification occurred using the steps of denaturation, annealing, and elongation at specific cycle times and temperatures. 95˚C was used for the denaturation step as it provided the correct temperature for the hydrogen bonds between the two DNA strands to break (Donohoe et al., 2000). To find the correct annealing temperature for the primers, multiple trials at various temperatures were necessary. The most successful temperature for the designed primers was found to be 51˚C, confirmed by multiple trials (Figure 2) (Figure 3). Extensions then occured as nucleotides were added to the new targeted DNA segment guided by the primer. To ensure an adequate concentration of the target zone in the PCR product, 30 PCR cycles were run for each trial. To possibly reduce primer dimer, the extension cycle time could be lowered, leaving less time for the primers to bind to themselves. Another strategy to reduce primer dimer could have been to decrease the primer concentration in the PCR cocktail (Etlik et al., 2008). This would have resulted in less excess primer remaining in the amplified product, creating less ghost bands the gels. A negative control was created to ensure that if results were not as expected, it was not rooted in the PCR cocktail, but in the agarose gel (Figure 5). Original annealing temperatures were predicted to be higher at 58.4˚C, but as research trials occurred, this was found to be too high and was lowered to improve specificity and band brightness.

Gel Electrophoresis

Gel electrophoresis was used to analyze the PCR products. It was found that a 1% agarose gel in a TBE buffer provided the best results. After first using .8% agarose gels, the 1% gel provided more distinct bands with less smearing. The higher agarose concentration allows the DNA bands to separate more completely than lower concentrations (Shiang et al., 1994). LB gels were also used to increase efficiency because of their shorter analysis time, but the 1 Kb plus DNA ladder and PCR products did not appear as prominently as in the TBE gels. The best results were achieved when the gel was run at a voltage of 120V. Although more efficient when run at higher voltages, portions of the TBE gel melted and results were compromised when run at voltages higher than 150V. Original predictions were that a .8% agarose gel would successfully present the bands created through electrophoresis but after multiple trials, 1% were ultimately used for better band separation and brightness.

DNA Extraction

To obtain wild type DNA, extraction from human IB3 cells was performed. After .08μg of DNA was extracted, the purity was found using spectroscopy. A purity value of 4.531 meant that pure DNA was extracted from the IB3 cells and could be used as a WT sample in the research assay. This purity value is significant to the research because if the DNA contains a higher concentration of proteins than nucleic acids, when run through PCR and gel electrophoresis the results could be altered. To further test the purity of the DNA, a sample was loaded into an agarose gel and run through electrophoresis. After viewing under ultraviolet light, the DNA remained in the well. This occurred because no primers were added to amplify a target zone and the DNA was pure to where no bands present in the gel (Reddy and Grewal, 2009). Mutant type DNA obtained from Coriell Cell Repositories had a purity value of 1.736 which was found using spectroscopy.

Social Simulation

To assess the psychosocial aspect of the G1138A mutation, a social simulation of the disease was performed. The difference in lifestyle and the struggles people with Achondroplasia face were easily detectable. To analyze the data collected, a linear regression was run on each severity level (Figure 6). This allowed researchers to connect severity to assistance needed. The linear regression generated from the recorded data validated the experiment and confirmed the original prediction that the mutation would cause great inconvenience. This inconvenience primarily resulted from the increased time necessary to perform the tasks. The two aspects of the simulation were social and psychological. When attempting to perform regular activities, assistance was often needed. This created a dependency that many people with Achondroplasia resent (Stevenson, 1957). To reduce the dependency of an Androplastic dwarf, grocery stores and libraries could make a greater effort to accommodate such mutations by having products available at multiple heights. This simulation was significant to the research assay, as it highlighted how a single point mutation on the fourth chromosome can significantly alter a person’s life.

Double Blind Experiment

The double blind experiment allowed researchers to prove that their research assay could be used to diagnose Achondroplasia. The ability to diagnose the mutation is important in science today with genetic screening. As more people begin prenatal screening for genetic mutations, efficient and accurate approaches are needed. The diagnostic assay shows that correctly designed allele-specific primers and PCR can be used for diagnosis. The mutated primers showed occasional annealing to the WT DNA, but the bands created were at base pairs less than 653, making the experiment flawed but viable (Figure 5). The validity of the experiment could have been further increased if multiple DNA samples were collected and also tested. This would increase the number of trials and number of variables, resulting in more results to compare and analyze error. Initial predictions did not consist of mutated primer annealing to wild type DNA, but ultimate conclusions were still drawn from the results.

Future Directions

Initial research was focused on finding a correct annealing temperature for PCR amplification. In several trials, no prominent bands appeared in the gel, signifying that the annealing temperature was incorrect. The best band produced was at an annealing temperature of 51°C. If additional time were allotted, half-temperature increments would be tested in multiple trials to determine the best annealing temperature for the primers. Improving primer design would also better the assay. The Yaku-Bonczyk designed mutant forward primer and the traditional reverse primer would be redesigned to decrease the amount of primer dimer and nonspecific binding in the gels. The mismatches at the 3’ end of the mutant forward Yaku primer may not successfully prevented annealing to the WT DNA. The reverse primer also had several nucleotide repeats, which allowed it to anneal at multiple locations. A possible sequence for the new reverse primer is 5’ – TCTGACTGGTGGCTG – 3’, creating a 667 base pair product. The extension time in PCR could also be reduced to 25 seconds to solve the primer dimer problem because there is less time for primers to bind to each other. The most significant improvement would be running a second double blind experiment. Wild type primers would be used in the experiment instead of mutant primers to decrease the amount of nonspecific binding that was produced by the mutant primers in the previous trial. In addition, other types of DNA would be tested in the assay besides the purified DNA from the human IB3 cells. This will increase the legitimacy and the usefulness of the experiment by decreasing smearing in the gel.