Diagnosis of Heterozygous CF Patients afflicted with

c.233dupT Mutation using Allele Specific PCR

 

                                   









By: Alisha Ungkuldee, Nicole Curtis, and Sara Kostecka









LB145 Cellular and Molecular Biology

Tuesday and Thursday 3 PM

Hayden Stoub and Marla Nazee

11/21/2017

 

 

 

Abstract

 

 

            The c.233dupT mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is a frameshift mutation found in primarily Hispanic subjects, causing a severe form of cystic fibrosis (CF) (Gregoire-Bottex and Soe, 2017). The addition of an extra thymine at the 233rd base pair on exon 3 of the CFTR gene produces a dysfunctional protein after translation, inhibiting chloride ion movement through epithelial cells (Gregoire-Bottex and Soe, 2017). Polymerase chain reactions (PCR) and gel electrophoresis were used to replicate and display the segment of interest of the CFTR gene in order to determine if DNA tested was wild-type or had the c.233dupT mutation. We hypothesize that primers designed with the Yaku-Bonczyk method for a diagnostic assay of the c.233dupT CFTR mutation will yield an adequate volume of amplified base pair products through PCR due to increased annealing precision by intentional nucleotide mismatches at the 3’ end. Our team utilized six primers including forward and reverse mutant primers from Johns Hopkins Lab, MD (Fprimer1 and Rprimer1), designed forward and reverse primers for the wild-type DNA allele (Fprimer2 and Rprimer2), and designed forward and reverse primers for the mutant DNA allele (Fprimer3 and Rprimer3). The annealing temperature for Fprimer1 and Rprimer1 was 52℃ for the first seven cycles and 68℃ for the remaining 23 cycles. An online theoretical annealing temperature calculator by IDT was utilized to calculate the annealing temperature for the designed primers; 54.7℃ for both Fprimer2 and Rprimer2 and 54.2℃ for both Fprimer3 and Rprimer3 (SantaLucia, 1998). We predict an amplified base pair product of 648 (base pairs 2641 to 3288) for Fprimer2 and Rprimer2 and 838 (base pairs 2641 to 3478) for Fprimer3 and Rprimer3 due to them being able to accurately anneal to the wild-type or mutant allele of the heterozygous DNA (Zielenski, 1990). The successful development of new c.233dupT annealing primers will allow physicians and scientists to progress the field of diagnosis and treatment of rare and understudied CFTR mutations in minorities.

 

Discussion

 

Experiment Summary

            Cystic fibrosis (CF) is an autosomal recessive disease caused by the mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on chromosome 7 which codes for the CFTR protein that facilitates chloride ion movement through epithelial cells (Welsh and Smith, 1995). When a mutation is present on the CFTR gene, the CFTR protein production malfunctions and chloride ion movement is inhibited (Rowe et al, 2005). This change causes thickened mucus in lumen and airways negatively impacting various organs within the body such as the lungs, liver, pancreas, and the small intestine (Welsh and Smith, 1995). In the case of the frameshift mutation c.233dupT, the CFTR protein is not produced due to the addition of a thymine at the 233rd base pair on exon 3 which causes a truncation (Hull et al, 1994). In our experiment, the question addressed is whether or not designed primers can be used in a PCR assay to correctly anneal and amplify to the c.233dupT DNA segment and further be used for diagnosis of the rare mutation through agarose gel electrophoresis. We hypothesize that primers designed with the Yaku-Bonczyk method for a diagnostic assay of the c.233dupT CFTR mutation will yield an adequate volume of amplified base pair products through PCR for diagnosis due to increased annealing precision by intentional nucleotide mismatches at the 3’ end causing there to be two nucleotide mismatches instead of a single mismatch that Taq polymerase could skip over (Yaku et al., 2008).

Original Predictions

            Using the purified wild-type DNA recovered from a team mate’s buccal cells and the c.233dupT heterozygous mutant DNA generously donated from the John Hopkins DNA Laboratory, MD, we predicted the lengths of amplified base pair products yielded from the PCR primers used for each DNA sample. Primers were designed using the Yaku-Bonczyk method where an intentional mismatched nucleotide is placed at the 3’ end to decrease annealing efficiency for a more precise annealing location (Yaku et al., 2008) (Dieffenbach et al, 1993).  Based on the hypothesis that the primers will correctly anneal from the mismatch at their 3’ end, and based on the locations of the primers on the genome, we predicted base pair products of 307 bp for the published primers (Fprimer1 & Rprimer1)(Taylor, et al., 1983), 648 bp for our designed wild-type primers (Fprimer2 & Rprimer2), and 838 bp for our designed mutant primers (Fprimer3 & Rprimer3)(Zielenski et al, 1990). For any samples where a wild-type primer was used in mutant DNA or vice versa, we predicted that the band would be unable to leave the well of the gel or stop at the top of the ladder due to the inability to properly anneal and being left with extremely large products (Chavanas et al., 1996). Since our mutant sample is heterozygous, we predicted each lane containing a PCR solution in its well would produce two bands; one band observed for the proper annealing of a primer to its respective allele (ex. mutant primer to mutant DNA allele) and one band remaining in the well of the gel for reasons previously stated (ex. Mutant primer to wild-type DNA allele) (Chavanas et al., 1996).

Results and Ultimate Findings

            In order to establish an effective PCR protocol, multiple experiments were run with variations in the PCR mixture concentrations, temperatures, and times as each factor greatly affects the annealing capability of the primers to the DNA template (Garibyan and Avashia, 2013).

In the first phase of the overall experiment, known Lambda bacteriophage virus primers and DNA template were utilized as a control in developing the PCR protocol to be used in the rest of the experiment. The base pair product yielded from agarose gel electrophoresis of Lambda was 348.28 bp; a percent error of 11.83% from the known 395 bp (Figure 4)(Taylor, et al., 1983). We predict the approximately 50 base pair difference was due to belated removal of the agarose gel from the Bio-Rad Powerpak electrophoresis machine which would allow for an elongated extension period of the ladder and PCR band essentially causing them to surpass the 395 bp mark (Garibyan and Avashia, 2013).  To achieve bands close to the target, multiple PCR protocols were researched and sampled from to develop an adequate mixture of ingredients and concentrations (see methods for concentration and ingredient details). Ultimately, the concentrations did not change throughout the experiment, however, we added the ingredient magnesium sulfate (MgSO4) as it assists with annealing (Mattila et al., 1991). The main factors in the PCR protocol that were altered were the annealing temperature and denaturation, annealing, and extension times. We started off with an annealing temperature of 58.8℃, calculated with an IDT annealing temperature estimator (see methods), which did not yield any amplified base pair products. Predicting that the adequate temperature would be within a few degrees of the software estimated temperature, we set up a temperature gradient over the next four trials ranging in temperature from 58.8℃ to 62℃ and found the best results, our 348.28 bp band, resulted from a temperature of 61℃. For the times, we originally began with a protocol of 45 seconds for denaturation, 30 seconds for annealing, and one minute 30 seconds for extension. We then altered our protocol to be 30 seconds, 60 seconds, and 90 seconds respectively to allow for a longer annealing period. After this change, we observed bands and continued to use this PCR protocol for any remaining PCR trials.

            Once clear control bands were achieved from Lambda, we moved into the next phase of our experiment that would also act as a second control. As stated earlier, John Hopkins provided heterozygous DNA for the PCR of their published primers and our designed primers. In order to further perfect our PCR protocol before testing our designed primers, we followed the protocol designed by John Hopkins as well as their primers to attempt to match their known amplified base pair product of 307 bp. We utilized the same PCR mixture we developed previously (see methods for details) but used their PCR protocol for times and temperatures. Their PCR protocol consisted of a total of 30 cycles with a change in annealing temperature from 52℃ for the first seven cycles to 68℃ for the remaining 23 cycles and 45 second intervals for each PCR stage (see methods for specifics). We replicated their protocol for multiple trials and only observed faint bands beneath the ladders which we predicted could be attributed to primer dimers (Brownie et al., 1997). Since primer dimers are circumvented most times by altering the annealing temperature (Brownie et al., 1997), after a few trials, we decided to alter their published protocol. We kept their original PCR protocol concerning temperatures and times, but we changed the annealing temperature to a gradient of a range from 45℃ to 51℃ (from IDT annealing temperature calculator) over five samples (five samples of wild-type and five of mutant). In these gels, we observed very faint bands in both the wells and down the gel as we originally predicted for the heterozygous DNA sample (Figures 5 & 6). Although the bands were faint in the mutant gel and there is evidence of primer dimer, we calculated a percent error of just 7.42% for the mutant allele band on the gel.

            From these findings, we can conclude that our amplified base pair predictions were correct and that the PCR protocol we developed could be used in further research of primers designed for the c.233dupT mutation. However, until further research is done, we were not able to support or refute our hypothesis regarding the Yaku-Bonczyk approach for designing primers.

Future Directions

              In order to be able to support or refute our hypothesis concerning Yaku-Bonczyk primer design, further experimentation would be required. In future experiments, we would alter our previous protocol for our published primer gels by creating a new temperature gradient in order to have more precise bands and to circumvent the possible primer dimers observed. We observed the most ideal bands closer to the 51℃ end of the gradient so the new gradient would be set between the temperatures of 51℃ to 61℃.

            Once clear, precise bands are observed on a new agarose gel, we will move into the final phase of our experiment. We will use the same PCR protocol developed for the Lambda and published control, but use primers designed using the Yaku-Bonczyk method. For the wild-type allele of the heterozygous DNA, we designed a forward primer (Fprimer2) that, at the 3’ end, consists of a cytosine which corresponds to the wild-type allele sequence (5’- GCC CTT CGG CGA TGT TTT ATC -3’). For the mutant allele of the heterozygous DNA, we designed a forward primer (Fprimer3) that, at the 3’ end, consists of an extra thymine due to the c.233dupT frameshift mutation (5’- GCC CTT CGG CGA TGT TTT ATT -3’). The reverse primer for the wild-type allele (Rprimer2; 5’-TTC CTC CTT GTT ATC CGG GTC-3’) was designed to anneal at a base pair location that would yield an amplified base pair product of 648 with Fprimer2. Conversely, the reverse primer for the mutant allele (Rprimer3; 3’- CCG GGG TAC CGT GTA TAT AAG -5’) was designed to yield a base pair product of 838.

            With successful amplification of the wild-type and mutant alleles at the c.233dupT loci verified by a semi-log plot, our hypothesis concerning the use of the Yaku-Bonczyk primer design method could be supported. From there, diagnostic assays using our designed primers for the c.233dupT CFTR mutation could be readily utilized by diagnostic laboratories.

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Figures

A                                                            B

https://lh5.googleusercontent.com/Hm22fyh1vjmBNdzXmqvTL-OKyPwG4qWgjV4HScepNaegBbW4NJYgYKa63kKOj56PcgZBxGb2LQtHvYZlZluoPt0UfcL8Nu811WgESRVUr52-V2eJGqIE7EzAPvs01fEj1LkbzpA- https://lh5.googleusercontent.com/GY2wTxeYAb33gOdaBtnDR7v75NHfrSJUI51S7MYOlLkO5Ykz2nY3wqo33Dwfh1J2lA_aFY0kpMMJNSqJudiyz66uYjWiw5_kA1NWuigdhOAfToYCdR-YEoVa3MpWxiKiJ7mvITD1



Figure 6. Agarose gel electrophoresis resulting from PCR of mutant published primers and heterozygous mutant DNA for c.233dupT. (A) Agarose gel was run at 200V for 11 minutes.  For the first and eighth well, a mix of 10 ΅l of Kb+ ladder and 2 ΅l of bromophenol blue dye was inserted to create a visible ladder to use as a point of reference for migration distance of bands. In the second well, a negative control of 10 ΅l nuclease-free water and 2 ΅l bromophenol blue dye were inserted. In the third through seventh well, 10 ΅l of the PCR cocktail containing heterozygous mutant DNA and the mutant primers, along with 2 ΅l of bromophenol blue dye were inserted. As expected, bands showed up in both the top wells and at 307 base pairs in lanes three through seven. This result shows that the wild-type allele of the mutant DNA was not amplified, while the mutant allele of the DNA was amplified due to successful annealing of the mutant published primers. (B) A semi-log plot was created in order to calculate the approximate molecular size (base pair length) of the acquired bands. Ladder values from Invitrogen were used in comparison to measured band distance from the wells in centimeters, especially for the 307 base pair band of interest. A power line was used which resulted in an equation of y=156201x-3.896 and an R2 of 0.97342.