Identification of the G551D-CFTR Mutation via PCR and Gel Electrophoresis

using Human Buccal cells

By: Ally Baur, Matthew Cederman, Maggie Leff, and Megan Monforton

LB 145 Cell and Molecular Biology

Tuesday 12:40-2:30 PM

Davin Hami, Emily Nemeth, and Ted Van Alst

1/29/2019

(Title page written by: B-888)

(Revised by: B-999)

(Finalized by: B-678)

Abstract

Written by: B-888

Revised by: B-999

Finalized by: B-678

The G551D mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is the third most common mutation and accounts for 3% of all Cystic Fibrosis patients (Bonnie et al., 2011). The mutation is a single base pair substitution in the 551^st codon that substitutes an amino acid from GGT (Glycine) to GAT (Aspartic Acid) (Bompadre et al., 2007). Allele specific Polymerase Chain Reaction (PCR) amplification with forward and reverse primers was used to determine the presence of the G551D mutation within one allele. A third nested primer and restriction endonuclease, Pme1 from New England Biolabs, will be used to verify the correct amplified region. We used the the intentional mismatch of the Yaku oligonucleotide design in our diagnostic assay for patients to increase specificity and decrease the amount of false positives encountered when looking for the existence of the G551D mutation (Yaku et al., 2008). The amplified segments were then evaluated through the use of agarose gel electrophoresis to look for the presence of the G551D mutation within the sample DNA by the detection of a single band 882 base pairs long when using the wild-type DNA with wild-type primers, same with mutant primers and mutant DNA (Zielenski et al., 1991). We will further evaluate the amplified region by using Pme1 and expecting a third primer to verify when using agarose gel electrophoresis. Through the development of this specific assay, doctors can use this technique to accurately screen and diagnose for G551D mutation.

Discussion

Written by: B-999

Revised by: B-888

Finalized by: B-699

Experiment Summary

Cystic Fibrosis (CF) is an autosomal recessive disorder that primarily affects the Caucasian population (Ernst, 2011). CF affects mainly the lungs, pancreas, reproductive organs, digestive tract, and sweat glands due to an imbalance in the regulation of chloride ions caused by defective CFTR proteins (Welsh and Smith, 1995). The G551D missense mutation is considered the most prevalent class three gating mutation with a worldwide prevalence of ~3% (Bompadre et al., 2007) and is most common in northwest and central Europe (Gaudelus, 2013). The CFTR protein consists of 27 exons. Located on exon 11 is the G551D missense mutation, caused by a single nucleotide base pair substitution from a guanine (G) to an adenine (A) at the base pair 107,844 within the CFTR gene (Zielenski et al., 1991). This alteration results in a glycine (GGT) to aspartic acid (GAT) at the 551st amino acid of the CFTR protein. The mutation causes a significant reduction in chloride ion flow across epithelial cell membranes because the mutation directly affects the NBD1 (nucleotide binding domain 1), resulting in the cystic fibrosis disease (Sermet-Gaudelus, 2013). The modified structural folding of the CFTR protein results in the inability for ATP to bind at the NBD1, which provides the required energy to unclog the regulatory domain from the CFTR ion channel (Sheppard and Welsh, 1999). This causes inability to open the channel of CFTR. Our research team is posed with answering the question of, can a specific diagnostic PCR assay be devised to accurately identify human genomes that contain the G551D-CFTR missense mutation? It is hypothesized that the implementation of an intentional mismatch utilizing the Yaku method located on the third base and a site specific mismatch on the first base from the 3’ end of our designed forward primers (107,815bp to 107,844bp in exon 11) will reduce false positives in our assay for the detection of G551D (Yaku et al., 2008). Followed by subsequent confirmations, third designed forward primer (108,066bp to 108,096bp in exon 11) (Kai et al., 1991) as well as the restriction enzyme Pme1 from New England Biolabs (108,500bp to 108,501bp), will increase the confidence and specificity of the genomic detection of the G551D-CFTR missense point mutation. The specificity will be increased due to the steric hindrance between the 3’ end of the primer and the DNA template, resulting in the inability for Taq to polymerize an unspecified DNA strand (Ayyadevara, 2000). Our confidence stems from the implementation of the secondary confirmations by amplifying/splicing at specifically known regions within the original target sequence. With the use of customary designed primers, followed by the third primer and restriction enzyme Pme 1, the application of our specific PCR assay would have a positive impact on the medical field by allowing doctors to have a greater confidence in their diagnosis and establish appropriate treatment more adequately.

In addition to development of our diagnostic PCR assay, we also collaborated with film making researchers in a 30 Days Experiment to document the progression of our lab group over the duration of the semester. The goal of this documentary experiment was for the filmmakers to observe our lab group’s improvement in collaboration, problem solving, and crucially transferable laboratory skills. This documentary provided useful and applicable evidence for understanding how researchers build a better foundation on scientific knowledge and protocol within a specifically designed lab.

Original Predictions

Using PCR and the Yaku primer method to design a set of primers, we will be able to amplify DNA responsible for producing part of the NBD1 of the CFTR protein from CF patients that will be analyzed through gel electrophoresis. We will use three different primers to administer our experiment. The two forward primers that we designed consist of forward primer wild-type 1 (FPWT1) and forward primer mutant-type 2 (FPMT2). In addition, we created a universal reverse primer 1 (URP1) that will anneal all DNA samples regardless of the status of the G551D mutation. We predicted that there will be successful annealing of the FPWT1 to the homozygous wild-type gene in the DNA sequence between base pairs 107,815 through 107,844 in exon 11. The addition of complementary base pairs through polymerization will occur because there is no steric hindrance between nucleotides of the wild-type DNA sequence and FPWT1 (Patel et al., 2001) along with the annealing of URP1 on base pairs 108,670 through 108,697. Additionally, we also predicted that the FPMT1 will anneal with the homozygous mutant DNA sequence in the same location as the FPWT1, resulting in elongation because there is no mismatch between the base pairs of the mutant DNA and mutant seeking primer. (Robertson et al., 1998). As a result of successful annealing, the predicated band result is 882 base-pairs because that is the length of the DNA sequence targeted by FPWT1 and FPMT1, and each of the two primers are designed to allow them to anneal to the wild-type DNA or mutant respectively (Lago et al., 2017). We predict the presence of no band will be a result of a mismatch between the last nucleotide of the 3’ end of the FPMT1 to the homozygous wild-type genomic sequence, or the FPWT1 to the homozygous mutant genomic sequence. This is because the steric hindrance between the nucleotide bases is too significant for Taq polymerase to bind and elongate (Patel et al., 2001).

Results and Ultimate Findings

The use of assessing Lambda DNA through PCR amplification and gel electrophoresis analysis serves as a positive control for our assay and supports that our PCR cocktail and methods are reliable. Our initial creation of an agarose gel and composition of PCR amplification through manual PCR yielded no identifiable DNA bands when the amplified target lambda DNA was analyzed. This negative result showed fault in our gel electrophoresis, attributing this to running the gel at 254V for ten minutes due to limited time. Making note of this error, we hypothesized that running the gel electrophoresis at a lower voltage for a longer time would produce better DNA bands under analysis. Because the gel provides the DNA fragments in both the PCR sample as well as the DNA ladder, more adequate amount of time would allow the bands to fully migrate properly (Southern, 1979). When the gel was ran at 200V, the 1kb DNA ladder appeared visible, resembling the predicted separation of the known band lengths (Figure 1). The migration distances of the DNA ladder were investigated and constructed into a graphical representation of base pair vs migration distance (cm). Utilizing this graph, the migration of the Lambda DNA band was assessed, and the migration distance corresponds to a DNA segment length of 385 bp (Figure 1). The theoretical length based off of the genomic sequence for Lambda DNA from New England Biolabs with our two primers, 1-Rz1F and 1-Rz1R, is 395 bp. We predict that this additional DNA migration is attributed to running the gel electrophoresis at too high of a voltage for too long, 200V for 20 minutes. We will troubleshoot this error by conducting gel electrophoresis at an even lower voltage and running it for a longer duration of time, resulting in more ample time to migrate and separate based on molecular weight. We adjusted our original PCR cocktail procedure and adjusted it to increase amount of Taq polymerase from 0.5µL to 1µL (Eckert and Kunkel, 1990) as well as increased the amount of dNTP’s from 1µL to 1.5µL (Roux, 1995). In addition to the cocktail alterations, we increased the number of PCR cycles as well as the denaturation and elongation times to ensure proper amplification. Noting these specific changes, the results observed show a DNA band produced in well 3 corresponding to the amplification of Lambda DNA (Figure 1). While casting the second agarose gel, the comb we utilized to create the wells punctured through the entire width of the gel unobserved until closer inspection after gel electrophoresis was conducted, resulting in no DNA band present within lane 5 (Figure 1). To further correct this, in agarose gels that will be casted in the future we will be manually propping the comb up in order to overt this situation again. Experimentation with custom wild-type designed primers, published primers, and our nested primer with purified human buccal cell DNA was ran after successful amplification of Lambda DNA. With adjusting and troubleshooting the errors mentioned, we were able to get results for wild-type primers with purified human buccal cell DNA, shown in wells 3 and 4 as faint bands (Figure 3) and band results with our nested primer in wells 2 and 3 (Figure 4). Additionally, results were analyzed after the use of published primers with purified human DNA in well 2 (Figure 2) as a very faint band at 426 bp. Bands for lambda DNA were present above the ladder after running out PCR with published primers and nested, which was predicted to be caused by the larger size of base pairs, preventing it from migrating through the gel. Possible errors that occurred for faint band results with wild-type primers and published primers are unpurified DNA prior to establishing a better genomic purification protocol (see methods) and improper annealing temperature. Centrifugation for a shorter period of time and only once may have contributed to these unsuccessful results. Fixing these errors resulted in brighter bands after running PCR with purified buccal cell DNA and our nested primer (Figure 4), as well as with our 3rd conformation primer (Figure 5). Another possible human error could’ve been flaws in the proper concentrations of water and DNA through pipette errors.

Future Direction

Overall factors such as improper annealing temperatures and concentrations of reactants in our PCR cocktail, such as water and DNA, affected the results of our bands analyzed through gel electrophoresis. By raising the annealing temperature with the Lambda DNA cocktails early on, forward and reverse primers were able to bind and function more optimally thus avoiding the issue of primer dimers, which occurred in our PCR where we got extra bands that weren’t expected. The annealing temperature was set at 52℃ originally then raised to higher temperature in the next experiments (56℃), leading to higher success in expected bands appearing (Figure 1). In our PCR with purified human buccal cells and published primers, there were no bands present when viewed in fluorescent light after the gel was run primarily. We predicted that this issue was rooted from improper DNA to water ratio as well as annealing temperature. We used 38.6µl of distilled water with .2µl of DNA which resulted in no bands. In the following PCR, we changed this by lowering the water and raising the DNA concentration (3 µl water, 4.7 µl DNA), which resulted in appearance of a very faint band in well 2 (Figure 2). For less error in the future, we predict that using even less water in the DNA cocktails will further improve the results of our bands, such as 5µl DNA and 2µl water. Off of these changes in annealing temperature and water to DNA ratio, we predict that if we had time for further experimentation with G551D mutant samples, we could confirm the success of our mutant-type primer. As a result of time constraints, we were not able to utilize Pme 1 restriction enzyme in our experiment. If further experimentation was implemented, Pme 1 would allows us to test the efficiency of our PCR assay and accuracy of targeted band amplification.

Figure 4: Implementing G551D-CFTR detection with FPWT1. (A) Well 1 was loaded with 7μL of 1kb+ ladder and 3μL of (10x) bromophenol blue gel loading dye. Wells 2-5 were loaded with PCR amplified DNA that was targeting the volumes of PCR DNA and deionized water utilized in a 10μL PCR reaction tube. Each PCR tube contained the following reactants in a 10μL reaction tube: 0.25μL FPWT1, 0.25μL URP1, 1μL 10x PCR buffer, 0.3μL MgCl₂ (25mM), 0.3μL Taq polymerase, 0.2μL dNTP’s (10mM), and the respective amounts of deionized water and purified human DNA as indicated above each well. These contents were micro-pipetted into a 0.5 mL microcentrifuge tube and placed into a thermocycler for 5 minutes of initial denaturation at 95℃ then 36 cycles of denaturation, annealing, and elongation. Denaturation occured at 95℃ for 30 seconds, followed by annealing at 62℃ for 1 minute, then elongation at 72℃ for 1:30 minutes. Afterwards, final elongation at 72℃ of 7 minutes. The resulting gel as analyzed and imaged to detect amplified DNA bands. (B) A semi log plot was created of the logarithm of the molecular size vs the migration distance. The measurements were based upon the migration of known molecular sized DNA bands in the 1 kb+ DNA ladder of well 1 and used to make the semi log plot. Utilizing the linear trendline equation of y = -0.2241x +4.2463, the amplification length of the DNA band in well 4 was found to be 822 base pairs; with an expected amplification length of 882 base pairs.