Polymerase chain reaction. A useful tool for analyzing DNA mutations, including mutations related to cystic fibrosis (CF) is polymerase chain reaction (PCR).  PCR reactions have been used in the past for many purposes; including identification viruses, genetic mutations, and mycobacterium (Taylor et al., 1997).  In PCR, annealing temperatures are crucial, especially to the G542X mutation since it is a one base pair deletion.  Primers may anneal non-specifically if the temperature is too low (Wittwer et al., 1993).  Knowledge of effective primers and annealing temperatures for the G542X mutation may give insight into more useful and streamlined methods of PCR (Wittwer et al., 1993). 

             Standard E. coli and lambda virus protocol. This experiment was intended for practice using PCR not related to G542X.  There were four trials performed using lambda viral DNA two times and E. coli bacterial DNA three times.  Changes made to reach success through four trials were the concentration of lambda DNA increased from 5 micrograms/mL to 100 micrograms/mL, lowering of the annealing temperature from 50°C to 49°C for E. coli, and reducing the time for denaturation, annealing, and extension steps from one minute to 30 seconds.  Increasing the amount of DNA allows a greater abundance of it for primers to anneal to, lowering the annealing temperature allowed binding of primers to the DNA, and decreasing the time for PCR reaction steps gave less time for non-specific binding (Wittwer et al., 1993).  Appearing on the gel were two bands for the lambda viral DNA at 500 base pairs and one for the E. coli at 530 base pairs.

            Genome purification. For genome isolation of S9 cells, Qiagen Kit with protocol for DNA purification from cultured cells and cell suspensions was used.  To confirm the presence of DNA, spectrometers were used to obtain a concentration of the DNA in the S9 sample.  Also used to confirm was standard gel electrophoresis of the DNA.  The 1XKB ladder was present, verifying the gel ran properly (Wright, et al. 2009).  Loaded into the gel was loading dye and purified DNA; loading dye was used to weigh down the DNA so it would run within the gel.  No bands appeared in the gel because primers were not used; instead there was glowing DNA at the well, under UV light.  The brighter the glow at the well indicates a greater abundance of DNA; the DNA will not leave the well because it is genomic, and is too heavy to run down the gel during electrophoresis (Wright et al., 2009).  

G542X Experimentation. PCR was used to isolate and amplify a nonsense mutation on exon 11 of the cystic fibrosis transmembrane regulator protein (CFTR) gene, known as G542X (Kerem, 1990).  It occurs at 1756 base pairs of the nucleotide sequence (Lefers, 2004).  The purpose of this experiment was to contribute designed primer sequences and PCR conditions for G542X mutation for the development of a multiplex screening test for cystic fibrosis. The mutant positive primers designed by the research team would anneal if the DNA was positive for the G542X mutation, resulting in a positive test, meaning the patient has the disease CF.  The wild-type positive primers would anneal if the DNA did not contain the G542X mutation.  It was hypothesized that G542X wild-type positive sequence would produce a band of 589 base pairs, in comparison to the 1XKB ladder, in the agarose gel when S9 DNA cells were analyzed.  The wild-type G542X band must appear in the agarose gel, through standard gel electrophoresis, to support the hypothesis (Wittwer et al., 1993). 

Primers were also designed to test for ∆F₅₀₈ because it is the most common mutation among CF patients (Wieczorek et al., 2008).  While the focus of the research was to develop ideal PCR conditions and primers for G542X mutation, ∆F₅₀₈ was also tested for to confirm the research group’s ability to design effective primers, and also to confirm the presence of heterozygous ∆F₅₀₈ in S9 DNA cells (Reiniger et al., 2005).  ∆F₅₀₈ wild-type and mutated primers were also designed to anneal to the DNA at 575 base pairs.  Therefore, it was also hypothesized that bands of 575 base pairs would anneal when mutated and wild-type ∆F₅₀₈ primers were used with S9 cells in standard gel electrophoresis.

Human error in experimentation includes but not limited to mistakes using the pipettes, spills of the reaction cocktail, contamination of the DNA sample, miscalculations of the annealing time and temperature, and poor programming of the PCR and electrophoresis machines.  If the PCR machine cannot regulate its own temperature or the annealing temperature is too high, the test will result in no amplification of the DNA region (Wright et al., 2009).  If annealing temperature is too low, non-specific binding can occur.  Mistakes when using pipettes include not setting the measurement correctly or using the same tip multiple times, leading to contamination or the wrong amount of solution drawn.  Contamination of solutions will make the reaction cocktail incorrect because there will be too much, or too little of a particular component. 

           Prenatal CF screening survey. The purpose of this experiment was also to supply survey data on opinion of prenatal screening for CF by a population with scientific background; it is important to know if scientists support genetic screening, so they will utilize and continue research on the multiplex CF screening test.   The information for prenatal screening will be gathered through online multiple choice question survey, using Surveymonkey.com; the link was sent to Lyman Briggs students in introductory level biology and chemistry courses.  The survey was given online to reach a greater number of people, and was the best way to reach those surveyed.  It was hypothesized that the majority of those surveyed would support prenatal screening for CF.  This belief is based on ten- year study which concluded about two thirds of people are willing to undergo prenatal genetic testing (Singer et al., 1998).  Explanations for the outcome include increased knowledge of the subject, scientific advances, and portrayal in the media. The data was analyzed through a chi-square test, which is a good statistical test for this type of information (Lydersen et. al., 2007).  The chi-square value found based on the surveyed data was 6.01; with a p-value of 0.9-.1 on the 78 surveyed population.  Based on this information calculated, the conclusion reached was that awareness of cystic fibrosis is independent of the people’s opinion on prenatal screening.  Approximately 63% of the population studied were in support of prenatal screening for CF, 33% have no opinion regarding prenatal screening, and 4% are not in support of prenatal screening.  Possible sources of error in this study include unintentional bias in the survey questions, insufficient survey responses, and a sample, made of mostly college students, does not adequately represent an entire population.  Future research conducted after the survey could study the correlation between a person’s political identity and his or her opinion of prenatal screening. Future research could also study how family members of people with genetic diseases feel about prenatal screening.

            The multiplex screening tests for multiple possible mutations of the CFTR protein at once (Smith and Welsh, 1995).  One team of scientists was able to use PCR to identify eight mutations of cystic fibrosis (Chehab and Wall, 1991). Information about the PCR conditions for G542X will be compiled with information about the PCR conditions for other mutations.  PCR conditions refer to annealing temperatures, lengths of time for each cycle, and the primers designed.  Including G542X in the screening test is especially important, since it is the second most common cystic fibrosis mutation (Levy, 2004). 

           If research on G542X continues future experiments can focus on finding ideal PCR conditions. Finding better conditions may help with not only screening, but possibly treatments (Thomas et al., 2009).  Geneticists may one day use the information about the inner-workings of DNA to design gene therapies that may help code for CFTR proteins in cystic fibrosis patients.