Yaku Primer Design Method for PCR Inconclusive in Identification of the G542X Mutation of CFTR in IB3 Cells

by:

Ashleigh Campbell, Kristyn Cubba, Kelsey DeLand, and Steve Lesner

LB 145: Cell and Molecular Biology

Monday 10:30 AM

David Malakauskas and David Maison

11-14-2009

Abstract

The G542X nonsense mutation of CFTR occurs when guanine is exchanged for thymine in the 542^nd amino acid, glycine, resulting in the creation of the stop codon GTA. This halts CFTR synthesis and results in the lethal disease cystic fibrosis (Loirat et al., 1997). IB3 DNA from CF patients was obtained, purified, and tested for G542X. It was predicted that the Yaku method of primer design will create a more specific genetic screening assay to test for the G542X mutation due to the two base pair mismatch on the first and third base pairs on the 3’ end of the primer. This should’ve ensured annealing to only the targeted area of the DNA (Yaku, et al., 2008). The PCR cycles were controlled using a thermocycler and the resulting amplified DNA was examined using agarose gel electrophoresis. The presence of bands containing 529 base pairs signifies the presence of the G542X mutation (Yaku, et al., 2008). Data collected and analyzed did not display conclusive results to support the hypothesis. A significant amount of non-specific binding and incorrect band were displayed. However, an agarose gel run at 40°C showed bands at the predicted band length using both the mutant and wildtype primers, indicating a heterozygous genotype. Surveys were conducted with several target groups to determine the opinions for using animal models for lethal diseases like CF. It was predicted that representatives of animal-focused professions will be opposed to the testing with animals because of their stereotypical sympathy toward the animals they work with, while individuals with a human medicine-based background will be in favor of the testing on animals in order to create cures for humans (Festing and Wilkinson, 2007). The data collected refutes our hypothesis: on the whole, veterinarians and science professors felt animal testing was more ethical than the medical professionals.

Discussion

Experiment Summary

Cystic fibrosis (CF) was at one time considered a childhood disease because the major physiological problems that accompanied the disorder did not allow those suffering from the disease to live past their teenage years (Hopkin 1998). Fortunately, in recent years, several new treatments have been developed to help manage the symptoms caused by CF, stretching the current life expectancy for CF patients to over 37 years (Couzin-Frankel, 2009). CF is caused by a mutation of the CFTR gene, which is located on chromosome seven. The focus mutation of this study, G452X, is caused when the amino acid glycine (nucleotide sequence GGA) is converted to a stop codon (nucleotide sequence GTA) (Kerem et. al. 1990). As a result of the G542X mutation, a Class I mutation occurs and the CFTR protein’s translation is halted in the cell, consequently causing a lack of chloride transport ability into and out of the affected cells.

PCR is often used to diagnose CF and other genetic mutations and there are several different methods that can be used to obtain results. One such method involves the implementation intentional mismatches, resulting in fewer to no non-specific binding, also known as the pseudo positive problem (Yaku et. al., 2008). For this experiment, two reverse Yaku-type discriminating primers were designed. Rprimer 1 was designed to anneal to wildtype DNA (despite one intentional base-pair mismatch) and to not anneal to mutant DNA because of two base-pair mismatches. Rprimer 2 was designed to anneal to mutant DNA (despite one intentional base-pair mismatch) and to not anneal to wildtype DNA because of two mismatches. A third, non-discriminating forward primer was designed to anneal to both types of DNA 529 base pairs from each reverse primer.

A sociological study was also performed to determine the opinions of several science-based professionals on animal testing. This study was modeled to reflect research done by Rogers et al (2009). In their research, scientists bred female CF heterozygote hogs to male CF heterozygote hogs, resulting in litters with the expected ratio of 1:2:1genotypes. The piglets from the resulting litters were studied in vivo from twelve to forty hours, and then euthanized and dissected (Rogers et. al., 2009). Research was conducted to determine whether scientific professionals from several backgrounds felt that it was ethical to induce a terminal disease on an animal with the intent of euthanizing the animal 40 hours after birth. Those surveyed included veterinary technicians and veterinarians, nurses, medical technicians, and doctors, and professors of science. Responses to each of the questions from each interviewee were coded using sociological techniques.

Original Predictions

It was hypothesized that the use of the Yaku method would result in a successful diagnosis of the G542X mutation due to two base-pair mismatches between the discriminating primer and the non-matching strand of DNA. The presence of a band of 529 base pairs on an agarose gel following electrophoresis would support correct binding of Rprimer 1 to wildtype DNA and Rprimer 2 to mutant DNA. Non-specific binding was not expected to occur because of two intentional mismatches on the 3’ end. Rprimer 1, when used in combination with Fprimer 1, was expected to anneal to wildtype DNA, and reverse primer 2, when used in combination with the forward primer, was expected to anneal only to mutant DNA. If the DNA sample was heterozygous for the G542X mutation, it was predicted that both primers would anneal. If no bands were present, it was possibly due to an error in calculating the annealing temperatures or due to a base mismatch, which would disrupt the extension phase of PCR (Haenisch et al, 2009).

In regards to the sociological survey on animal testing, it was predicted that veterinarians and veterinary technicians would be most opposed to the breeding of animals with lethal diseases, concurrent with the beginning of the veterinarian’s oath, which states that veterinarians will use their skills to protect animal health and relieve animal suffering (Feldmann and Carding, 1973). However, it is possible that there may be some movement away from this prediction, as the second part of the oath states that veterinarians must also work for the promotion of public health (Feldmann and Carding, 1973). It was additionally predicted that medical doctors, nurses, and medical technicians would be in favor of animal testing, as animal testing is used to promote human medical research and is often part of the process by which treatments are approved for human use. Additionally, a study performed in the United Kingdom found that 96 percent of general practitioners accept the use of animal testing as ethical (Festing and Wilkinson, 2007). However, a study performed in India in 2006 found that most medical students were opposed to animal testing, although they did feel that animal models were necessary for their comprehension of medicine (Dhingra et al, 2006). It was predicted that the opinions of science, math, sociology, philosophy, and history professors at Lyman Briggs College, Michigan State University would fall somewhere in between the opinions of veterinary and human medical professionals, as a result of their diverse academic backgrounds.

Ultimate Findings

Various experimental methods were used in attempt to determine the presence of the G542X mutation. The first five gels varied in annealing temperatures. These gels were run at temperatures between 37˚C and 44˚C: the most specific products were found at temperatures of 40˚C and 41˚C. From here, the buffers used in each PCR cocktail were altered. Each buffer contained levels of Tris HCl, ammonium sulfate, potassium chloride, magnesium chloride, and Triton X-100: for exact values, refer to the methods section. These buffers did not end up providing a significant change in results obtained: as a result, salt levels were altered. By altering the concentration of magnesium chloride, it was believed a more specific product would become present. However, the lengths of the bands found to be present in the gels still were not of desired length. Non-specific binding was still found to be prevalent. After various steps in trouble-shooting, it was determined that from here cycling times should be extended. Denaturation, annealing, and extension temperatures were each extended by 15 seconds, for a total time of 45 seconds each. The final extension time was doubled as well, to 10 minutes in length. Also, the number of cycles ran increased from 30 cycles to 35. No additional significant results were obtained. Over the course of the experiment, 24 agarose gels representing over 50 different cocktails or annealing temperatures were run, with a definite band of a target length of 529 base pairs not achieved. In comparison of the conventional method of primer design versus Yaku primer design, the conventional method provided a band of targeted length on the first trial.

One hundred and eighty professionals from the fields of veterinary medicine, human medicine, and scientific professors were interviewed to determine their opinions regarding the ethics of animal testing. The results of the surveys provided several insights into the realm of animal testing: their responses to nearly all questions failed to support the hypothesis. All of the science professors and veterinary professionals interviewed felt that animal testing was an ethical method for research on disease, while one medical professional did not, showing a deviation from our predictions (Table 1). All professionals, regardless of their affiliation, felt that the accumulation for cures of diseases would decrease if animal testing were banned, showing that while some may not agree with animal testing, they understand the role it plays in finding cures and treatments for diseases. Veterinary professionals felt that results obtained through animal testing were no more legitimate than results obtained without using animal testing, while all others had the opinion that animal testing legitimized the findings of research. All professionals that felt animal testing was an ethical method for research were in agreement that it was ethical to induce or breed an animal with a lethal genetic disease: some even expressed opinions that this is the only type of research that should be performed on animals, as it is necessary to increase the lifespan of the human race. Lastly, nearly every professional had the opinion that it was no more ethical to perform testing on mice than on pigs, showing that anatomical similarities do not necessarily harbor more sympathy towards a certain species: however, when asked if there are some species that should not be used for animal testing, some indicated that primates and humans should not be considered for possible testing subjects, suggesting that enough anatomical similarity invokes feelings of sympathy. Four p-values were found to be less than 0.05. A p-value less than 0.05 shows that the data obtained from the question could be considered significant. The questions that were found to be significant, which are listed in the appendix, are #3, #4, #5, and #6.

Future Directions

The sociological animal testing survey indicated interesting results that provide several venues for future research. Previous to this study, there weren’t any studies analyzing how the opinions of professionals in the medical and veterinary fields compared. Due to time and financial restraints, surveys of these groups could only be conducted within Michigan State University and a few other isolated areas in Michigan. It would be interesting to not only compare opinions nation-wide, but globally as well to determine whether there are discrepancies of opinion between the two professions.

References

Brewer, N.R. 1999. Architectonics of laboratory animal science. 50 Years of Laboratory Animal Science. American Association for Animal Laboratory Science, Memphis. 7-11.

Campbell, N.A. and J.B. Reese. 2007. Biology-8^th ed., Chapter 14 “Mendel and the Gene Idea.” Benjamin Cummings, CA.

Dhingra, M.S., Singh, A., and Singh, J. 2006. Animal experiments and pharmacology teaching at medical schools in India: a student’s eye view. Alternatives to Animal Testing and Experimentation 11(3): 185-191.

Feldmann, B.M. and Carding, T.H. 1973. Free-roaming urban pets. Health Services Reports. 88(10): 956-962.

Festing, S. and Wilkinson, R. 2007. The ethics of animal research. European Molecular Biology Organization Reports 8(6). 526-530.

Haenisch, et. al. 2009. LB145 Course Pack. MSU Printing Services, East Fee Hall. Michigan State University, East Lansing, MI.

Hopkin, K. 1998.Understanding Cystic Fibrosis. University Press of Mississippi, Jackson.

Kerem, B., Rommens, J.M, Buchanana, J.J., Markewicz, D., Cox, T.K., Chakravarti, A., Buchwald, M., and Tsui, L.C. 1989. Identification of the Cystic Fibrosis Gene: Genetic Analysis. Science. 4922(245): 1073-1080.

Lerer, I., M. Sagi, G.R. Cutting, and D. Abeliovich. 1992. Cystic fibrosis mutations AF508 and G542X in Jewish patients. J Med Genet 29: 131-133.

Loirat, F., S. Hazout, and G. Lucotte. 1997. G542X as a probable Phoenician cystic fibrosis mutation. Human Biology 69(3):419-425.

Newton, C.R., and A. Graham. PCR, Second Edition. Springer-Verlag Hong Kong Ltd., Hong Kong.

Patterson, J.M. 1985. Critical factors affecting family compliance with home treatment for children with cystic fibrosis. Family Relations 34(1): 79-89.

Qiagen Inc. 2007. Protocol: DNA Purification from Culture Cells and Cell Suspensions. Generation Capture Column Handbook. 17-19.

Rowe, S.M., S. Miller, and E.J. Sorscher. 2005. Cystic Fibrosis. New England Journal of Medicine 352: 1992-2001.

Rabinow, P. 1996. Making PCR: A Story of Biotechnology. The University of Chicago Press, Chicago.

Rogers, C.S. et al, 2008. Disruptions of the CTFR gene produces a model of cystic fibrosis in newborn pigs. Science 321: 1837 –1841.

Welsh, M.J. and A.E. Smith.1995. Cystic Fibrosis. Scientific American: 52-59.

Yankaskas, J.R. and G.B. Mallory, Jr. 1998. Lung transplantation in Cystic Fibrosis. Chest. 113:217-226.

Yaku, H., T. Yukimasa, S. Nakano, N. Sugimoto, and H. Oka. 2008. Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem. Electrophoresis 29: 4130-4140.

Figures and Tables

Figure 1: Gel electrophoresis analysis of band lengths using annealing temperatures of 39 and 40°C in analysis of the G542X mutation in IB3 cells. The cocktail used for this reaction included: 29 mL of H₂O, 5 mL 1X LB buffer, 10 mL dNTPs, 2 mL forward and reverse primer, 1 mL DNA template and 1 mL Taq polymerase. The PCR cycling included a 5 minute initial denaturation at 94°C; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 39 or 40°C for 30 seconds and extension at 72°C for 30 seconds; followed by 5 minutes of final extension at 72°C. The ladder was analyzed using a semi-log plot. All lanes of the gel displayed a significant amount of non-specific binding and dark spotting of the dye in the beginning and end of the lanes. Lanes 5 and 6 show clearer bands with less non-specific binding than lanes 2 and 3. Lane 5 displayed bands at 526 and 344 base pairs (bp); lane 7 displayed a band at 456 bp; lane 2 displayed bands at 1213 and 526 bp; and lane 3 displayed a band at 744 bp. The desired band length was 529 bp. In attempt more precise band lengths, the annealing temperature of the samples was raised to 41 and 42°C.

Panel A

Panel B

Figure 2: Average coded response values from animal testing survey filtered by profession (panel A) and gender (panel B). 180 individuals were involved in the survey in total: 130 from the veterinary field, 20 from the medical field, and 30 science professors. Additionally, of all participants in the survey, 130 were female and 50 were male. Answers for questions 1 and 2 were not coded with a numerical value, as they were used to determine the individual’s gender and profession: question 13 was omitted as it involved a free-form response. The responses to all of the questions displayed above, with the exception of question 6, were coded on a scale of 0 to 3, while responses to question 6 were coded on a scale of 0 to 2. The questions and their respective response codes can be found on the last two pages of the methods section.