Unsuccessful Detection of G542X on CFTR using PCR and Gel Electrophoresis Coupled with Survey Analysis for Cystic Fibrosis Research Funding

Written by: Greg Capoccia

Revised by: Shae Valko

Finalized by: Rachel Baywol, Greg Capoccia, and Shae Valko

by: Rachel Baywol, Greg Capoccia, and Shae Valko

LB 145

Thursday 7:00 to 10:00

Marci Baranski and Josh Olszewicz

April 30, 2009

Abstract

Written by: Greg Capoccia

Revised by: Shae Valko

Finalized by: Rachel Baywol, Greg Capoccia, and Shae Valko

Our research set out testing the hypothesis that an assay could be designed for efficient identification of the cystic fibrosis nonsense mutation, G542X, using Polymerase Chain Reaction (PCR) and electrophoresis. PCR was performed in four separate reaction tubes containing an ingredient cocktail, each of which contained two of three primers specifically tailored to the normal wild-type (reverse; #1), a locus 800-bp away from mutation (forward; #2), and the mutant gene (reverse; #3). Amplified PCR products were run through agarose gels by electrophoresis so to observe the presence (or absence) of the G542X mutation by confirming the presence of mutant DNA. From the allele-specific amplification research of Wittwer et al. using wild-type and mutant primers, to the conclusion of Saiki et al. that PCR enriches amplification by 10⁶, and the work of Vogelstein and Kinzler using PCR to identify cystic fibrosis in a minor fraction of cells, we predicted that positive band readings of 800 base pairs would occur when forward and wild-type primers annealed to the wild-type DNA, and when forward and mutant primers annealed to mutant G542X DNA. Mutant DNA was not acquired within the timeline of this experiment, thus it could not be tested. No positive bands were observed during PCR when running wild-type DNA with forward and wild-type primers, refuting our hypothesis. A sociological survey was given out to test our hypothesis that a survey could be conducted to reveal a potential correlation between science and non-science majors, and their opinions on the allocation of cystic fibrosis funding. Base on previous suvery analysis of hepatitis B, we predicted that science majors would devote funding amongst cystic fibrosis patients, while non-science majors would appropriate funding exclusively to ∆F508 (Wilson et al., 2001). Analysis of findings revealed that 63% of those surveyed preferred exclusive ΔF508 funding, refuting the hypothesized science major position, while supporting that of non-science majors. The larger goal of our research involves working with other research groups to design an assay for detecting multiple CFTR mutations.

Discussion

Written by: Shae Valko

Revised by: Greg Capoccia

Finalized by: Rachel Baywol

Future Studies Section by: Rachel Baywol, Greg Capoccia, and Shae Valko

The G542X mutation is derived from a nucleotide change of G to T resulting in a stop codon that replaces glycine as the 542nd amino acid on exon 11 in the CFTR protein sequence, thus truncating it (Alibakhshi et al., 2008). Since it occurs in approximately 2.4% of cystic fibrosis patients, G542X has been identified as the second most prevalent of all known mutations (Alibakhshi et al., 2008; Du et al., 2002; Loriat et al., 1997). Thus, the importance to provide an efficient test for identification of G542X lies in the number of patients afflicted by this nonsense mutation.

The intent of this research was to develop an efficient method for identification of the G542X premature stop mutation in cystic fibrosis patients. Given this, the research hypothesized that a test could successfully be designed to detect the G542X mutation on the CFTR gene. This assay involved the extraction of DNA from IB-3 cells (wild-type), and then testing both mutant and wild-type DNA samples as templates in PCR with specifically designed primers. Of the three primers, two reverse primers were created: one that would exclusively anneal to the G542X point mutation on the CFTR sequence, while the other would anneal when wild-type DNA was templated during PCR. Subsequent agarose gel electrophoresis of the PCR mixture would identify whether the G542X mutation is present in the DNA. It was predicted, based on the research of Wittwer et al. and the use of primers tailored for allele-specific amplification, that after gel electrophoresis, bands would be seen at approximately 800 base pairs in both wild-type and mutant PCR assays due to the design of our primers – there are 800 base pairs between the 5’ end of forward primer 2 and nucleotide position 1756, the 5’ end of reverse primers 1 and 3.

DNA Extraction

The presence of a faint band observed when the isolated IB3 bronchial epithelia

cell DNA underwent gel electrophoresis indicated a base pair length of more than 4100-bp. The appearance of faint band potentially indicated an impure DNA isolation product that contained RNA and/or proteins (Amita et al., 2002; Moss and Solomon, 1991). While ultimately this product would be undesirable for efficient PCR, this fact was ignored and used as the wild-type DNA template in the four PCR trials conducted.

PCR and Agarose Gel Electrophoresis

Of the four PCR and subsequent gel electrophoresis trials using the impure IB-3 wild-type DNA extraction, none resulted in positive bands. Gel analysis, however, did provide some indication as to what may have procedurally gone wrong in certain trials. Troubleshooting techniques were developed in response to each gel, and applied to the subsequent PCR trials.

Initial PCR of the 25 µL reaction cocktail contained 13.5 µL nuclease-free water, 1µL Taq polymerase, 2.5 µL buffer, 5 µl dNTP, and 1 µL for each primer (1 and 2) stock solution (2 µL primer, 198 µL nuclease-free water); it was annealed at a temperature of 52˚C. Under UV light, this PCR product resulted in no visible bands on the agarose gel.

PCR trial two maintained the same cocktail ingredients, while the annealing temperature was lowered to 46˚C. The lower annealing temperature was based on the prediction that the 52˚C annealing temperature used in PCR trial one was too high to allow bonds to form between the primer and denatured DNA (Hecker and Roux, 1996). The loaded gel now received a reduced voltage supply of 80 V with the intent of preventing any genomic drifting between lanes as lower voltage application retards DNA movement towards the positive anode (Görg et al., 1999). Gel analysis revealed neither bands nor observable gel irregularities.

With no bands observed in the two previous trials, troubleshooting led to a change in the cocktail solution of trial three. The primer stock solutions (primers 1 and 2) were diluted so each stock had a larger concentration of pure primer: stock solutions to contain 5µL pure primer and 195µL nuclease-free water; the higher concentration of primers would potentially increase primer annealing (Czerny, 1996). Since no visible genomic degradation was observed when the annealing temperature was lowered in trial two, the annealing temperature of 46˚C was maintained in trial three. With a lower voltage in trial two yielding no bands, the voltage was returned to the 132 V.

Analysis of gel three observed no bands within the DNA ladder, however, below the ladder the presence of faint bands for all six lanes was visible. These bands indicate that primers had annealed to the each other rather than the denatured DNA – primer dimers (Brownie et al., 1997). Primer dimers occurred due to the high primer concentration in the cocktail, leading to weak interactions between primers: the high concentration of primers increase the likelihood that primers would anneal to one another (Brownie et al., 1997).

The presence of primer dimers led to changes in the reaction cocktail for trial four: to compensate for the high primer concentration, the volume of wild-type DNA was increased (Brownie et al., 1997). Two different cocktails maintaining the primer stock solution from trial three were created: the first increased the volume of DNA to 2µL, while the volume of nuclease-free water was lowered to 12.5µL; the second contained 3µL of DNA and 11.5µL of nuclease-free water. Gel analysis of PCR trial four observed no bands whatsoever on the gel.

Potentially the nonexistence of bands in all four trials can be attributed to the impurity of isolated DNA during genomic purification. While the spectrophotometer was initially used to test

Cystic Fibrosis Funding Survey (DIY)

While no definitive results were observed from the PCR assay, the proposed DIY experimentation produced more promising findings. The DIY involved a sociological issue surrounding funding for cystic fibrosis. A survey was composed hypothesizing a potential correlation between science and non-science majors, and their opinions on cystic fibrosis funding. The survey was given to both science and non-science majors attending Michigan State University, and asked whether the students thought cystic fibrosis research funding should be strictly given to the DF508 mutation (being the most prevalent) or if it should be equally distributed among mutations. Based on research protocol and mode of data analysis for hepatitis B funding survey conducted by Wilson et al., it was predicted in using a survey similar to theirs that students with a science major would advocate funding equally amongst the mutations, while non-science majors would devote funding exclusively for DF508 (-Wilson et al., 2001).

Analysis of survey results indicated that 37% of science majors, and 26% of non-science majors favored exclusive funding towards DF508, as was originally predicted; while 12% of science majors, and 15% of non-science majors favored equal distribution of funding amongst all mutations. The remaining 10% (4% science and 6% non-science) could be attributed to those students who did not believe any funding should go towards cystic fibrosis research. This could imply that those people did not understand the questions asked. One subject (a Human Biology major) ranked his prior CF knowledge as a 5 on a scale from 1 to 5 and ranked the severity of CF as a 10, the highest level on the survey. However, this subject responded with a “no” for both ΔF508 funding and equal funding distribution. There is also the possibility that this small percentage of subjects simply do not find CF serious enough a disorder to deserve funding.

These findings refuted the hypothesis generated at the beginning of the investigation given that the minority of science majors favored equal allotment of funding for cystic fibrosis research

Given that the hypothesis can be refuted, this could have been explained by the fact that funding of diseases from the National Institute of Health directly relates to the societal burden of that disease (Gross et al., 1999). Taking the Gross et al. conclusion into account, it would have been more probable to predict that the surveyed Michigan State students would have advocated the allocation of cystic fibrosis funding entirely to ∆F508.

Future Direction

Due to lack of success in this particular research, several variables should be taken into account if more time was allotted for the research of an assay to efficiently identify the G542X mutation using PCR and agarose gel electrophoresis. One possible adjustment to PCR would involve prolonging the annealing phase of PCR: as opposed to a 35 second per cycle annealing phase, 45 seconds to a minute per cycle would be allowed to maximize the possibility for primers anneal to any DNA in the PCR cocktail. Another variable that should be taken into account for future research involves carefully designing primers to minimize the potential for primer dimers (Brownie et al., 1997). Given that primers were designed under the supervision and assistance of David Malakaukas, it is rather unlikely that this would be necessary; regardless, the possibility could exist.

As discussed previously, several issues encountered during this assay involved impure DNA and improper mixing of PCR cocktails. In the future, it is imperative that a spectrophotometer be used to ensure DNA purity. The ideal A260:A280 ratio is about 1.7 (Kehrmeyer et al., 1996; Moss and Solomon, 1991; Suarez-Martinez et al., 2006). Keeping this number in mind will help certify more satisfying PCR results that would contain traces of RNA and/or proteins (Moss and Solomon, 1991). To make sure PCR cocktails are more properly mixed in the future, a micropipette would be used to create the homogenous mixture necessary for optimal PCR outcomes.

For the DIY portion of this investigation, several changes would be acknowledged. First, a larger sample size would be taken into account for future study. While the number of people surveyed in this experiment was relatively small (only 98 subjects), in the future more people could be questioned in order to receive more accurate, non-biased data. Another adaptation that could be inspected involves surveying a broader diversity of people. Rather than dividing only students into science and non-science majors, more specific majors could be outlined. Also, the opinions of more professors and grad students could be taken into account.

If this future research would ultimately support the hypothesis that a PCR-based gel electrophoresis identification, and the predictions coupled with it were met, one means of further substantiating the success would be through the use of restriction enzyme digestion. Furthermore, with a potentially successful identification of mutations via gel electrophoresis and PCR with future research, a much more economical PCR identification test could be constructed that would encompass reaction cocktails containing a number of primers tailored to specifically anneal to one of a number of mutations. This would ideally allow any cystic fibrosis inflicted patient of an unknown mutation to be tested not only for allele specific amplification via gel electrophoresis, but also to confirm the presence of a mutation as evidenced by the base pair length dictated by the primers.

Figures

Written by: Rachel Baywol

Revised by: Rachel Baywol, Greg Capoccia, and Shae Valko

Finalized by: Greg Capoccia

Figure 4. Observation of primer dimers in the gel electrophoresis for PCR product diluted for higher concentration annealing at 46˚C: Trial Three. In observance of no bands or any other indications for specific troubleshooting in trial two, the dilution of the primer stock solutions was altered for a greater primer concentration: each 1µL stock solution for both forward and wild-type reverse primers were composed of 5µL primer and 195µL nuclease-free water; all other reaction cocktail ingredients remained constant as in trials one and two. The absence genomic degradation in the PCR trial two gel resulted in maintaining the annealing temperature at 46˚C. After PCR, the gel was loaded with two ladder dyes at the very opposite lanes of the gel, while 10µL PCR product/loading dye mixture were individually loaded into those lanes marked “Lane 1” to “Lane 6.” Given the perceived lack of success running the gel at 80V, the trial three gel was supplied a constant of 132V as in trial one. After 20 minutes, the gel was placed under UV light exposing no bands within the ladder base pair length region, rather below the ladder region, six faint bands were observed under each lane. These faint bands, also known as primer dimers, were the result of the forward and wild-type reverse primers annealing to one another, instead of denatured DNA. As indicated by Brownie et al., primer dimers can be correlated to the higher concentration of primers, where the increased presence of primers results in weak interactions between the forward and reverse primer sequences as opposed to DNA. The subsequent trial would see an increase in DNA template volume to potentially counteract and eliminate primer dimers.

Figure 6: Cystic Fibrosis funding survey taken by 98 Michigan State Students with the intent of studying a relation between non-science and science major responses. Analysis of our surveys showed that most students generally preferred the majority of funding be provided to ΔF508 research. A greater percentage of science majors indicated a preference for funding ΔF508 research as opposed to favoring equal fund distribution, as was originally predicted. Another rather interesting finding was that 10% of those surveyed (4% science and 6% non-science) responded “no” to both exclusive ΔF508 funding and equal funding distribution. This could imply that those people did not understand the questions asked. One subject (a Human Biology major) ranked his prior CF knowledge as a 5 on a scale from 1 to 5 and ranked the severity of CF as a 10, the highest level on the survey. However, this subject responded with a “no” for both ΔF508 funding and equal funding distribution. There is also the possibility that this small percentage of subjects simply do not find CF serious enough a disorder to deserve funding.