PCR and Gel Electrophoretic Analysis of Canine Cells to Reveal R1456W and R812W Missense Mutations on CFTR Gene

By: Caleigh Griffin, Katy Kesler, Greg Ribble, and Ahmad Tahawi

www.msu.edu/~griff475

http://youtu.be/L6IHqP5hDB4

Abstract

Mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) are commonly associated with cystic fibrosis (CF) (Welsh and Smith, 1995). The purpose of this experiment is to identify mutations within the CFTR gene in the Canis familiaris species. Through the attempt to identify the presence of the R812W or R1456W mutations, it was hypothesized that mutant-seeking forward primers will anneal to only the mutant DNA during PCR because of the intentional mismatch in the primer design and will thus reveal a band via gel electrophoresis (Spadafora et al, 2010; Yaku et al, 2008). In order to ensure the accuracy of the equipment and protocol, primers designed for the Lambda virus Rz gene were tested prior to testing the designed primers. Successful amplification of the forward and reverse primers for the Rz gene amplified a 495 bp band that was consistent with the expected amplification. Published control primers were also obtained from Spadafora and his research group for the amplification of exon 27 (320bp), which resulted in a successful amplification of 322bp (Spadafora et al, 2010). For the experimental primers, two forward primers were designed (a mutant and wild-type) to reveal the presence or absence of either the R812W or R1456W mutation. DNA was amplified using PCR and evaluated using gel electrophoresis. The R1456W primer was designed to amplify 1003bp, and resulted in successful amplification of 972bp, while the R812W primer was designed to amplify 1013bp and resulted in successful amplification of 949bp during the experiment. To gain a better understanding of CF in canines, anxiety levels of the researcher and canine were studied after walking with and without wearing an Elizabethan Cone. Anxiety in the canine was determined primarily using heart rate, which indicated a significant difference (p<0.0001) between wearing and not wearing the cone. Anxiety levels within the researcher were primarily determined using a GAD-7 test, which resulted in a significant difference (p<0.0001) before and after wearing the cone. The experimental and socio-psychological results from this research provide for a better understanding of canine CFTR genotypes, and moreover attempt to further the research in discovering non-human models for CF (Spadafora et al, 2010).

Discussion

Experiment Summary

Cystic fibrosis (CF) is an autosomal recessive disease that is among the most common genetic disorders (Rowe et. al, 2005; Welsh and Smith, 1995). CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which was first discovered in 1989. Since then over a thousand mutations have been identified (Whittaker, 2007). The CFTR gene codes for the CFTR protein, where the wild-type acts as sodium and chloride channel on the apical membrane of epithelial cells (Rowe et. al, 2005; Welsh and Smith, 1995). Moreover, a mutation in the CFTR gene results in complications with the exchange of sodium and chloride in and out of the epithelial cells. This ultimately affects multiple organs in humans such as the lungs, liver, pancreas, small intestine, reproductive tract, and the skin. CF due to a CFTR mutation has never been identified in non-humans (Spadafora et al, 2010). However, since canines have been known to present symptoms similar to CF (such as pancreatitis and bronchiectasis), researchers have attempted to further investigate the existence of CF in species like Canis familiaris (Spadafora et al, 2010). This experiment focused on two of the missense mutations that were investigated by Spadafora and his group: R1456W and R812W. The R1456W mutation involves a single base pair substitution, replacing the 4366th base pair in the 1456th amino acid with a Thymine rather than a Cytosine. This changes the amino acid from an Arginine into a Tryptophan. The R1456W mutation decreases the CFTR function to approximately 20% of norm (Spadafora et al, 2010). The R812W mutation follows an identical substitution to the R1456W, however the substitution occurs on the 2434th base pair in the 812th amino acid. The R812W mutation causes a malfunction in protein kinase A, ultimately causing the CFTR channel to remain closed (Spadafora et al, 2010). In order to detect the R1456W and R812W mutations, polymerase chain reaction (PCR) was used in order to amplify the Canis familiaris DNA. A diagnostic assay was designed with PCR and gel electrophoresis in order to detect for the presence of R1456W and R812W mutations in the CFTR gene in canines. The resulting targeted DNA sequence from PCR will be deciphered by means of gel electrophoresis. Four forward primers were designed to detect the presence of the R1456W and R812W mutations: a wild-type primer for both the R1456W and R812W, and a mutant primer specific to the each mutation as well. Additionally, two reverse primers were designed to anneal 1003 and 1013 base pairs away from the R1456W and the R812W mutations respectively. In addition, control primers were taken from previous research done by Spadafora and his group, because their primers annealed correctly in their research (Spadafora et al, 2010). Ultimately, it was predicted that the use of PCR and gel electrophoresis could be considered a viable method for detection of the R1456W and/or R812W mutations because when primers are designed to anneal specifically to a mutation, given the right conditions the DNA should amplify where the mutation is present in order to produce a band on the agarose gel (Freeman, 2011). In addition to the PCR assay, an extension to the research was established by conducting a socio- psychological experiment to experience what it is like care for a canine diagnosed with a CFTR mutation. The socio-psychological experiment was conducted across a span of 30 days, and involved the measuring of anxiety levels of canines and the researchers in response to wearing an Elizabethan Cone during a walk at one of three random locations around Michigan State University’s campus. It was predicted that canines would experience heightened levels of anxiety after each walk where the dog is wearing the cone, because canines find comfort in their daily routines (Sherman et al, 2008). It was predicted that researchers would experience heightened levels of anxiety after each walk where the dog is wearing the cone, because of the human-animal bond that exists between a person and their dog (Sherman et al, 2008). It was predicted that canines would experience heightened levels of anxiety after each walk where the researcher is wearing the cone, because of the human-animal bond that exists between a person and their dog (Sherman et al, 2008). It was also predicted that researchers would experience heightened levels of anxiety after each walk where the researcher is wearing the cone, because human CF patients have been known to exhibit anxiety (Cruz et al., 2009). The overall analysis of the data collected was performed in an ANOVA test. In each of our tests, the calculated p- value was p<0.0001which showed that the heart rates and GAD-7 test scores were all statistically significant. Our prediction that canines would experience heightened levels of anxiety after each walk where the dog is wearing the cone was supported, because canines find comfort in their daily routines (Sherman et al, 2008). Our prediction that researchers would experience heightened levels of anxiety after each walk where the dog is wearing the cone was supported, because of the human-animal bond that exists between a person and their dog (Sherman et al, 2008). Our prediction that canines would experience heightened levels of anxiety after each walk where the researcher is wearing the cone was supported, because of the human-animal bond that exists between a person and their dog (Sherman et al, 2008). Our prediction that researchers would experience heightened levels of anxiety after each walk where the researcher is wearing the cone was supported, because human CF patients have been known to exhibit anxiety (Cruz et al., 2009).

Original Predictions

Genomic Purification

It was predicted that the Qiagen DNA purification would yield between 3 and 8μgs of DNA because when 1x106 cells are lysed and their DNA is purified and collected this was determined to be the theoretical yield. The theoretical yield helped to understand how much DNA was extracted follow the DNA purification.

PCR

It was predicted that successful amplification of the targeted DNA sequence through PCR would indicate the presence or lack of presence of the R812W and the R1456W mutations because analysis of the targeted DNA sequence using gel electrophoresis should show the presence of the mutation by displaying bands of 1013bp and 1003bp, respectively, when gel electrophoresis is used and read correctly (Spadafora et al, 2010). A combination of the wild- type primer with the wild-type DNA was predicted to correctly anneal and replicate the targeted DNA sequence because of the design of the primers, which was based off of Yaku and his group’s method of primer design (Yaku et al, 2008). Likewise, when the mutant primer was combined with DNA containing the mutation, the primer was predicted to correctly anneal and replicate the target DNA, this is also because of the method of primer design developed by Yaku and his group (Yaku et al, 2008). Both forward primers used the same reverse primer. Although the prediction is the same between finding the R1456W and R812W mutations, it is important to note that the set of wild-type forward, mutant forward, and reverse primers are different for each mutation (see methods).

Gel Electrophoresis

Correct amplification between the forward and reverse primers in the analysis of R812W was expected to show a band at 1003 base pairs, while correct amplification of the R1456W mutation is expected to show a band of 1013 base pairs. It was predicted that if PCR fails to amplify the targeted DNA sequence due to a disagreement between the primer and the DNA, then this would result in no bands because incorrect annealing would fail to amplify the targeted DNA sequence, leaving the gel electrophoresis with nothing to express (Yaku et al, 2008). As predicted, mutant primers would only anneal and show a band when ran against the mutant DNA, while the wild-type primers would only anneal and show a band when ran against the wild-type DNA. In order to ensure the accuracy of our predictions and visibility of the bands, the Yaku method of designing primers was used (Yaku et al, 2008). The Yaku method essentially requires for an intentional mismatch of the 3rd nucleotide away from the 3’ end of the primer, which is designed to never anneal to the complementary DNA in order to prevent from getting false positives or negatives when running PCR (Yaku et al, 2011)

Ultimate Findings

It was concluded that more experimental tests would have to be done in order to receive viable bands for the R1456W designed primers once ran in gel electrophoresis. The control primers bound to the DNA sequence and amplified a DNA sequence 320 bp long. Designed primers for the R812W mutation annealed and amplified a DNA sequence of 949bp long. Due to a need for more experimental tests, an assay that identified and diagnosed the R812W and R1456W mutations within the canine genome is unknown. For genomic purification, the DNA yielded 3.17 µg with a purity of 1.899. In terms of the canine anxiety and depression experiment, the average GAD-7 score during the control walk was 6.8,3 while the anxiety while the researcher was wearing the cone raised the GAD-7 scores to 12.66. After running an ANOVA test, the p-value was found to be p<0.0001 which shows that the data from the measured GAD-7 test scores is statistically significant. The average heart rate for the canine during the control walk was found to be 65.42bpm, while the average heart rate of the canine after wearing the Elizabethan Cone during the walk was 148.83bpm. After running an ANOVA test, the p-value was found to be p<0.0001 which shows that the data from the measured heart rates is statistically significant.

Future Directions

To improve this experiment for the future, more mutations would be tested for using PCR and gel electrophoresis. The identification of cystic fibrosis in species other than humans is still a relatively new discovery, thus needs to be developed in relating the findings given with the PCR and gel electrophoresis to the actual symptoms of the animals, or in this case, canines. To further investigate cystic fibrosis in nonhuman species, such as canines, more time would need to be supplied. By having additional time, PCR could correctly diagnose the R812W or R1456W mutation with the canine genome. It was predicted that the R1456W designed primers produced a controversial bands, which resulted in a smear within the gel electrophoresis because the primers contained an 88% C-G ratio instead of the recommended 40-60% ratio. A possible solution to having a high C-G content would be to administer the Hot Start PCR protocol. It is predicted that Hot Start PCR will prevent nonspecific binding because the Taq polymerase and primers are not mixed at the low temperatures where nonspecific binding occurs (Strien et al, 2013). Another way to re-design this assay would be to add betaine to the PCR cocktail. By adding betaine to the PCR cocktail, the C-G hydrogen bonds are weakened, resulting in more thorough denaturation (Henke et al, 1997). Another possibility would be to raise the annealing temperatures when placed in the thermocycler. The low annealing temperatures allow for nonspecific binding because the DNA would be unable to fully break all hydrogen bonds. In terms of the canine anxiety and depression experiment, it could take place over a period longer than 30 days allowing for more data collection. Larger sample sizes collected from different parts of campus rather than staying within three locations will maximize the volume and randomness of data collected, minimizing the effects of random statistical outliers, and decreasing the p-value therefore making it more statistically significant.

Figures

Figure 1