The Newtons

 

Genotypic Detection of G551D Mutation for Cystic Fibrosis in Human IB3-1 Cells Using Yaku Designed PCR Primers

 

 

 

 

 

 

 

By: Jake Lehman, Miles Kamaloski, Allison Riley and Laura Drotar

 

 

 

 

 

 

 

 

 

LB 145 Cell and Molecular Biology

3:00-5:00 PM

Eric Konotwicz and Joe Conley

11/23/2013

 

 

 

Abstract

 

G551D is a point mutation caused by a  nucleotide switch of adenine to guanine on nucleotide 1784, which is found on amino acid 551 (Gadsby and Nairn 1999). This is responsible for 5% of cystic fibrosis cases (Rowe et. al 2011). This mutation causes nucleotide binding domain 1 (NBD1) to remain unresponsive to ATP and the channel remains closed (Gadsby and Nairn 1999). Genotypic diagnosis of this mutation is important for proper treatment of cystic fibrosis. The purpose of this study was to create a better genotypic assay to more accurately diagnose the G551D mutation in patients affected by Cystic Fibrosis. It was hypothesized with the use of custom Yaku primers containing an intentional mismatch on the 3Õ end will more accurately diagnose those afflicted with the mutation in a blind study with  a reduction in false positives (Yaku et. al 2006).

Through the use of control primers from Scobie et al., bands containing a length of 285 base pairs were discovered via gel electrophoresis, indicating healthy DNA was successfully amplified and present (Scobie et. al 1996). Custom primers, one of which would anneal to an intron on the mutant DNA, were improperly paired with cDNA, which did not contain any introns. Due to this mistake, when gel electrophoresis was performed, multiple bands from 100 to 500 base pairs in length appeared. It appeared that the Yaku primer annealed to the exon and extended to the end of cDNA strands of different lengths (Wagner 2002). Through these results, it is suggested that the Yaku custom primer can anneal to mutant G551D DNA since  exon 11 is present in both cDNA and genomic DNA. In the future, it will be necessary to use these primers with genomic DNA rather than cDNA to confirm their efficacy. Additionally, a blind study should be done to prove the advantage of the custom primers versus others.

 

Discussion

 

Experiment Summary

The CFTR gene, located on chromosome 7, encodes for the cystic fibrosis transmembrane regulator protein. As little as one base pair mutation on this gene can cause Cystic Fibrosis.  This autosomal recessive disease is caused by a malfunction of the CFTR protein (Gadsby and Nairn 1999). Cystic fibrosis has profound effects on chloride channels in the epithelial membrane leading to major issues with the pancreas, lungs and intestinal tract. (Sachdeva et al. 2012) The G551D mutation is one of the 1600+ possible mutations that cause cystic fibrosis (Rowe et. al 2011) and it is due to a single base pair mutation at nucleotide 1784 in exon 11. This Glycine to Aspartate amino acid (Comer et. al 2009) change causes gating of the subsequent CFTR protein. It is hypothesized in the literature that this protein hydrolyzes in response to ATP at one-tenth the rate of a normally functioning protein (Gadsby and Nairn  1999). Since cystic fibrosis can be caused by a plethora of mutations, genetic testing with PCR is important in understanding and correcting the underlying problems rather than the symptoms (Liu et. al 1992).  With the creation of an accurate and reliable PCR assay of the G551D mutation we can identify its presence (Friedman et al. 1991).

Original Predictions

 

             As a control, the lambda virus was replicated in order to confirm the functionality of all components involved in the PCR process.  It was predicted that bands 400 base pairs in length would be seen with gel electrophoresis when ran next to a Kb1 Plus ladder (Powell et. al 1994).  Upon successful replication of the lambda virus, it was verified that all materials were fully functioning, and any accountable error in future experimentation would not be influenced by "faultyÓ materials.

After extracting DNA from wild type and G551D afflicted cells, allele specific PCR primers would be used to amplify the target region with the 3Õ end corresponding to nucleotide 1784. With the use of a mutant and wild type primer, the presence or lack of the G551D mutation would be determined by means of gel electrophoresis. With control primers from the literature as well as their protocol, it was expected that a band of 285 base pairs in length would be seen with proper amplification of wild type DNA (Scobie et. al 1996).

In addition to the two primers obtained from Scobie et. al 1996, another set of two custom primers containing intentional mismatches on the 3Õ end would be used to better discriminate mutant and wild DNA. These intentional mismatches would reduce the chance of a false positive result (Yaku et. al 2006).It was predicted that if the custom primers anneal correctly, a band of 181 base pairs in length would be found in mutant affected DNA (Kramer and Coen, 2001).After confirming both custom and control primers worked, it was predicted that a significant difference in the ability to properly detect the mutation would be seen in a blind study (Yaku et. al 2006).

Ultimate Findings

Through running PCR with the lambda virus, it was verified that all PCR materials were working correctly (Figure 1). Afterwards, the control primer experiment replicating the work of Scobie et. al was done to amplify wild type DNA. By being able to successfully replicate Scobie et. alÕs work, a optimal PCR cocktail and thermocycler conditions was discovered which could be used with the custom primers. Bands of 269 base pairs were seen on gel electrophoresis for the control primers (Scobie et al 1996) confirming the presence or absence of wild type DNA (Figure 2). Custom primers were designed using the Yaku method, in which an intentional nucleotide mismatch was included on the 3Õ end (Figure 3). Through this process, it was predicted that less false positives would appear in the PCR/electrophoresis process when compared to standard designed primers. In order to use these custom primers and knowing that the control primers were working, cDNA containing the G551D mutation was extracted from expression vector pcDNA 3.1 and was then ready to be amplified. The cDNA was ran through the Epoch Micro-Volume Spectrophotometer System, however the results received calculated that the DNA concentration was approximately .55µg/mL, which appeared to be much less than expected. Nonetheless, PCR was still carried out with primers to verify if the DNA would replicate.

            After running the custom primers with the G551D mutant DNA,  four distinct bands appeared between 100 and 500 base pairs in length when analyzed via gel electrophoresis (Figure 5). Up until this point, the fact that the mutant DNA was actually complementary DNA, or cDNA for short, was overlooked. cDNA, which previously had its introns spliced from the genetic sequence, would not have an annealing region for one of the custom primers. This spliced version of DNA, which was much shorter than its complete DNA counterpart,  had an actual length just over 5000 base pairs (Invitrogen 2010). The mutation which was then implemented into the cDNA via directed mutagenesis by Bompadre et. al.

Realizing this error helped explain issues that arose during the custom primer experiment. The first error pertained to UV spectroscopy, in which a low yield of DNA (20 times less than expected) was determined. After further analysis, it was determined that since cDNA is much smaller than original DNA, the UV spectrometer was not able to detect the miniscule cDNA strands as easily. As a result, false numbers may have been recovered from the spectrometer due to its inability to register the comparatively short molecules of DNA.

In addition to explaining the poor UV spectroscopy results, the fact that cDNA was used also explains why multiple bands  were seen on gel electrophoresis (Figure 5) . Since all introns were removed in cDNA, only the custom yaku primer which targeted an exon was annealing. The reverse common primer on the other hand, was located on an intron, and most likely did not anneal at all, due to no annealing site being present. Furthermore, all cDNA is not the same length (Wagner 2002).  It appears that multiple bands are the result of the custom primer annealing and extending to the ends of their corresponding cDNA strands until they ran off. When these strands of cDNA are different lengths,  the resulting amplified regions seen on gel electrophoresis are different (Wagner 2002). Since these distinct banding regions consistently occurred over 11 reactions, it was determined that the issue could not be due to either non-specific binding or primer dimers (Kramer and Coen 2001).

While a blind study could not be carried out with cDNA, the results are still promising. Since distinct bands were seen with cDNA amplification, it was apparent that the custom yaku primers which anneal at an exon region contained within cDNA, were properly annealing. Taking into note that the custom primers with intentional mismatches on the 3Õ end do indeed work, further work should be done with them.

Being able to accurately and effectively diagnose the G551D mutation is of importance in medical treatment of the underlying causes of cystic fibrosis (Liu et al. 1992). We hope that the creation of these custom primers can assist in this diagnosis process. The current primers listed in the literature have been employed for more than twenty years and the creation of a better, more effective primer may be of importance (Scobie et. al 1996). Nonetheless, a multi primer kit, testing for a wide range of cystic fibrosis mutations accurately and effectively is the end goal. Being able to take a sample of a patients DNA, run PCR and amplify ten or even hundreds of the most common mutations with one test would be of utmost importance. Using optimized primers in these kits would further the ability of medical practitioners to correctly diagnose patients. We hope that our primers are the first step in achieving this ultimate goal that will provide those afflicted with cystic fibrosis an accurate plan of treatment based on their genotype.

Future Experimentation

First, many of the results gathered from control experiments need refining. While distinct bands of amplification were seen, better, more robust results could be acquired. To improve this, electrophoresis should be ran with the products an additional time with accompaniment of a lower voltage. Through this process, bands that appear should become crisper and more defined with less streaking. If necessary, PCR should also be redone with a more refined gradient around 63.1¡C should be used in order to determine if a superior PCR product that can be generated. Such results would also pave the way for an improved PCR protocol with custom primers which follow a similar reaction to those in the control. With custom primers operational it may be possible to notice a significant difference in detection accuracy will be seen as compared to those mentioned in Scobie et. al. due to a reduction in non-specific binding and increased GC content (Yaku et. al 2006).

Continuing on this idea, the efficacy of the custom primers can be further tested with actual genomic DNA. The completed research suggest that custom Yaku primers containing intentional mismatches on the 3Õ can in fact be used to anneal with the G551D mutation. Performing an experiment with actual DNA and amplifying known regions of DNA, including introns, will be of importance in proving their effectiveness.

 To statistically prove this effectiveness,  a blind study should be performed to determine whether any significant difference can be noted between the custom primers and those used in Scobie et. al. Such results may lead to the creation of a better genotypic assay for those afflicted with cystic fibrosis in the future.

 

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Figure 2 - Control Primer Amplification of G551D with PCR: In this figure, PCR was ran with wild type DNA and control primers used in the work of Scobie et. al.(A) Each PCR tube contained a 50 µL total cocktail consisting of .5 µL Taq Polymerase, .2 µL each primer, 1 µL  dNTPs, 5.5µL custom buffer (containing 2.5 µL PCR buffer, .5 µL of Triton, 2.5 µL of MgCl2, and 9.3*10-5g of K), 2.5 µL DNA template, and 40.3 µL of nuclease free water. All contents of cocktail were first combined into a 550 µL master mix, in which contained all the above contents. The DNA used was acquired from patient IB3-1 cells given by DR. Douglas Luckie at Michigan State University. The IB3-1 cells contained dF508 and W1282X mutations, and were extracted with an efficiency of 7.95µg/mL, as calculated from UV light spectroscopy observation.7 wells of DNA were ran consisting of a gradient ranging from 65.2 to 50 ¡C. Specific annealing temperatures have been provided with their corresponding wells in the above figure. 1 Kb Plus ladders were added to both sides of the wells. The PCR process ran consisted of an initial denaturing phase of 5 minutes at 95 ¡C, followed by 22 cycles consisting of a primary denaturation of 94 ¡C for 1 minute, an annealing with corresponding temperatures of 30 seconds, and an extension period of 30 seconds at 72 ¡C. Gel electrophoresis was completed using an 100 mL .8% agarose gel in TBE (Tris-Borate-EDTA), with the inclusion of 2µL GloGreen dye. The electrophoresis process was done at 150V and lasted approximately 20 minutes. Each well was loaded with 8 µL of DNA and 2 µL of a 6x loading dye, consisting of .25% xylene cyanol, .25% bromophenol blue, and glycerol.  The best annealing temperature through analysis appeared to be at either 63.1 ¡C with good result and little streaking. After observation through UV light, a semi logarithmic  plot was made comparing the migration distances of the 1Kb Plus ladders compared to their corresponding base pair lengths (B). After doing so, the DNA migration distance was measured and a migration distance of 6.85cm was found. From this, the ladder semi logarithmic equation was applied to calculate that the bands recovered were of 269 base pairs in length. These results recovered indicate that the predicted base pair length of 285 base pairs was accomplished.

 

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Figure 5 - Custom Yaku Primer PCR in G551D Mutant cDNA:In this figure, G551D mutant cDNA was ran through PCR and gel electrophoresis with the accompaniment of Yaku designed custom primers. A 550µL master mix was created which was then derived into 50µL PCR tubes. Each PCR tube contained 5.5µL of custom PCR buffer (consisting of 10 mM Tric-HCl pH 9.0, 50 mM KCl, 2.5 mM MgCl2, and 1% Triton X-100) 0.5µL of Taq Polymerase (1 active unit), 1µ of 100mM DNTPs, .2µL of each 100mM primer, 4.5µL cDNA (0.55µg/mL), and 38.29µL of nuclease free water. PCR was ran with an initial denaturing phase of 5 minutes at 95¡C, followed by 21 cycles of PCR. Each cycle consisted of a 94¡C denaturing for 1 minute, followed by an annealing phase containing a temperature gradient from 50-60¡C for 30 seconds, and finally a 72¡C extension period for 30 seconds. After all cycles were completed, a final extension phase was ran at 72¡C for 10 minutes. Multiple bands are noted from 100 to 500 base pairs in length. These bands appear to be caused by inconsistencies in the length of cDNA (Wagner 2002). These bands suggest that the Yaku Custom primer, which binds to the G551D mutation on exon 11 is running to the end of the cDNA strand since it is not possible for the reverse primer to bind to cDNA. The reverse primer binds to an intron, which are not present in cDNA. These results suggest that the Yaku custom primer is working and annealing to the correct site. Further testing with genomic DNA and the custom primers should be done for further verification.