Abstract
Cystic Fibrosis is an autosomal recessive genetic disorder that often results in chronic lung infections, the driving force behind high CF mortality rates (Welsh, 1995). As there are thousands of known mutations on the CFTR gene, the ability to detect a specific mutation for diagnosing a patient is highly valued. E60X is a class I mutation that involves a Thymine substitution for Guanine at base pair 29086 (Duguˇpˇroux, 2004). We engineered primers for PCR to detect CF in a DNA sample, and whether or not the E60X mutation was present. Our hypothesis stated that primers 1 and 2 would bind to the DNA in the presence of the E60X mutation, and that primers 2 and 3 would bind only in wild-type CF DNA; through agarose gel electrophoresis we predicted an 800 bp band would be present. While CF patients seek treatment through antibiotics, recent studies found inefficiency in antibiotic cycles for CF patients who developed a biofilm (Stewart, 1995). On a more basic level, other studies have provided rationale to support the notion that antibiotic abuse is increasing in general, leading to antibiotic-resistant stands of bacteria in people (Mulvey, 2009). By designing a questionnaire pertaining to antibiotic use, we were able to collect data between 200 Lyman Briggs students and 200 university students for analysis through a Wilcoxon Signed-Rank Test, which we found a significant variation between the two groups abusive tendencies towards antibiotics (P-value=.022). Due to improper engineering, our primers were unsuccessful in binding where expected; agarose gel electrophoresis displayed two bands at 500bp and 800bp.
Discussion
Cystic Fibrosis is a disorder that involves mutations on the CFTR gene, which is located on chromosome seven; a deletion or substitution of nucleotide bases has been found to be the cause of this disease. One of the main problems associated with this is the accumulation of mucus within the lungs due to the depravation of chloride. A person who has Cystic Fibrosis will have malfunctioning epithelial tissues due to irregular Cystic Fibrosis Transmembrane Regulators that block chloride ions from entering the cell (Welsh, 1995). These malfunctioning CFTR membrane-spanning proteins inevitably cause pulmonary infections, accounting for 90% of the disability and death in persons with cystic fibrosis (Rowe, 2005).
In our experiment, the E60X mutation was selected for study. E60X is categorized as a class I mutation because translation is prevented by substitution of a Guanine nucleotide in place of a Thymine nucleotide on base pair #29086. The G-T substitution results in the sequence to code for a stop codon (TAG) in place of the glutamic acid (GAG), thus hindering the proper folding of the CTFR protein prior it exiting the nucleus for translation (Rowe, 2005). Our experiment was designed to determine whether or not the E60X mutation was present in a patient with Cystic Fibrosis. We hypothesized that our primers 1 and 2 would bind after PCR and the agarose gel would reveal a band at 800 base pairs to indicate that the E60X mutation was present in the provided DNA (Malone et al, 1991).
Using PCR, a large amount of provided DNA was amplified. To do this however a multi-step process was required. First, the reaction cocktail was created. This mixture contained various buffers, dNTPÕs, primers and template DNA. Upon completion, the PCR cocktail was subjected to a thermo-cycler, where it rapidly cycled through three different temperatures for extended periods of time. We predicted that primers 1 and 2 would bind under the circumstances that the DNA used was previously known to have the E60X mutation.
However, our experiment yielded neither positive nor negative results due to the extremely high concentration of our primers or too low of annealing temperatures. Our first gel exhibited non-specific binding and after diluting our primers to a 1:10 concentration with water a second gel was run which exhibited no bands at all. Upon investigation it was discovered that the primers were too diluted prohibiting any type of binding from occurring. Due to the high dilution there were not enough amplified segments in the gel to produce a band. While some were present, it was not enough to cause a band to be present in the agarose gel. In a third gel the primers were diluted to a 1:1 concentration, two bands appearing at 500 and 800 base pairs were present. In a positive test result only one band should be present at 800 bp; upon further research too low of annealing temperatures were found to possibly have been the result of multiple bands. After various other gels were run at increased annealing temperatures the same results was seen. From there, it was determined that the error was from the primers, as further examination towards the subject allowed certainty. Our primers 1 and 3 were improperly engineered due to the use of the anti-sense strand rather than the sense strands. This caused the primers to bind and replicate in opposite directions amplifying everything except our intended section of DNA.
For our antibiotic abuse portion of the lab, we distributed a questionnaire to a total of 400 students in order to compare the abusive patterns towards antibiotics of university students with Lyman Briggs students. We chose antibiotic abuse because broad-spectrum antibiotics are used to help with the symptoms of cystic fibrosis (Welsh, 1995). A random number generator was used to designate specific buildings to give questionnaires to university students, while Lyman Briggs students were surveyed in Holmes Hall. The questionnaire consisted of eight multiple-choice questions, each set of answers having an assigned point value corresponding to the level of abuse (Mulvey, 2009). A range was set to categorize each individualÕs abusiveness towards antibiotics based off their cumulated points after taking the questionnaire. A graphical representation of each groupsÕ distribution was then created to visually compare the students. In order to statistically analyze our data, we used a Wilcoxon Signed Rank Test to conclude whether the results were significant or not. We obtained a P-value of .022, thus indicating that our experiment was statistically significant. Generally, Lyman Briggs students have less abusive tendencies towards antibiotics than that of University Students.
If further studies were to be conducted, we believe it should be towards combining various primers that test for a wide range of mutations in cystic fibrosis. As there are over a thousand different mutations, finding a way to scan for a majority of them in one PCR would be highly efficient for determining which mutation is present in the patient. Given an additional six weeks to research, primers could be designed from the sense strand of each individual exon that will only bind if the exon is mutation free. Given that there are 24 exons in a CFTR gene typically only one or two of these are mutated, so being able to pinpoint which exon is mutated would greatly narrow the field of possible mutations. From here it can be determined which mutation it actually is based on research. Possible errors with these further studies could include the length of the primers being too long resulting in non-specific binding; this would also mean the annealing temperature would be very high. A possible solution to this could be breaking the exon into two or three parts and testing them all separately.
Figure
Gels showing non-specific binding
Gels showing non-specific binding. In seven of the gels ran, there were multiple bands throughout the DNA wells. In the top gel we used an annealing temperature of 44 degrees Celsius and we did not dilute our primers at all. Well one was the kb ladder, which allows us to measure where the bands are present. Well two had primers 1 and 2 along with water. Wells three had primers 2 and 3 along with water. Well four contained primers 1 and 2 and DNA from a cystic fibrosis patients. Well five contained primers 2 and 3 along with DNA from a cystic fibrosis patient. In well five you can see there are many bands present, we believe this is due to the fact that our annealing temperature was too low and our primers were much to concentrated.
The bottom gel shows a zoomed in view of the non-specific binding. The far left lane (lane one) is the kb ladder. Lane two was empty. Lane three contained primers 2 and 3 and DNA from a CF patient. Lane four contained primers 1 and 2 with the CF patientÕs DNA as well. The DNA cocktails in this were ran through PCR at a 48 degree C annealing temperature and had a 1:1 DNA dilution with water.
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