Identification of Mutation 1078delT in

the CFTR Gene in S9 epithelial cell from a CF patient Using Allele Specific PCR

By

Kortnee Calkins, Jim Makinen, Ben Marengo & Val Nauta

LB 145 Cell and Molecular Biology

Monday 7pm-10pm

Ashley Coulter and Katie Oleski

Written on: 9/14/09

Revised on: 10/05/2009

Written By: A40172438

Revised By: A40332010

Finalized By: A40525550

Abstract

Written By: A40172438

Revised By: A40332010

Finalized By: A40525550

The 1078 del T mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein is most common in Celtic populations in Brittany, France (Mullier et al., 1994). It is a deletion of one thymine base pair at the 1078 position on exon 7 of the CFTR DNA (Dorval et al., 1994). The deletion of the thymine base pair results in a frame shift (Zielinski, 2000). Allele-specific polymerase chain reaction will be used to amplify targeted DNA in order to determine if the 1078 del T mutation is present in Cystic Fibrosis Patients. Because of the frame shift, wildtype DNA will vary from mutant DNA downstream from the mutation site. Using this discrepancy, forward primers were designed to bind to only one of these variants of DNA. Therefore, we hypothesize that wildtype DNA will only be amplified using the wildtype primer (Forward Primer #2) and mutant DNA will only be amplified using the mutant primer (Forward Primer #1). This is because primers will not anneal to non-complimentary DNA (Urbance, et al., 2009), which is the case with Forward Primer #1 and wildtype DNA or Forward Primer #2 and mutant DNA. The DNA template used came from IB3 and S9 cells, which were both wildtype for 1078 del T. The goal is to use this discrepancy, along with allele-specific PCR and gel electrophoresis to create a diagnostic assay for the presence or absence of the 1078 del T mutation. Our research was inconclusive, and the assay ultimately deemed non-diagnostic due to the presence of non-specific binding after gel-electrophoresis. Because more than one band was present, we cannot conclude the band at 400bp was due to proper primer annealing and amplification. Findings from this research can help design better and more efficient genetic testing for patients with Cystic Fibrosis and other genetic diseases. In addition, interviews with a genetic counselor and respiratory therapist and addition research were conducted in order to gauge the sociological and psychological factors associated with CF. Our research suggests that sociological impacts of CF have a strong interplay with scientific research, both morally and financially.

Discussion

Written By: A40332010

Revised By: A40172438

Finalized by: A40703106

Experimental Summary

Cystic Fibrosis is the most common recessive disease in Caucasian populations, occurring 1 in every 2500 births (Moullier et al 1994). This genetic disease occurs when there is a mutation in the cystic fibrosis transmembrane conductance regulator, a chloride channel on the apical surface of epithelial cells. With a mutation present, chloride ions cannot move out the cell. This inhibits the movement of solute, and makes mucus thick and more susceptible to bacteria and infections, mainly in the lungs and pancreas (Welsh and Smith, 1995). The most common mutation is ∆F508, where three base pairs are deleted from the genome (Welsh and Smith, 1995). Our group investigated the mutation 1078 del T, which is the second most frequent in the French region of Brittany. It occurs in the about 4.96% of cases of cystic fibrosis in this region. This mutation is located on exon 7, and it occurs from a deletion of one Thymine (Dorval et al 1994). This mutation causes a frame shift, and makes 1078 del T a class one mutation (Rowe et al 2005). Using the correct primers, PCR cocktail, primer annealing temperatures, and lab techniques, we predict that our mutant primer will fail when used on the S9 cells of a cystic fibrosis patient due to the fact that DF508 is the most common mutation, and that our wild type primer will succeed (Zielinski, 2000). This will be detected using allele-specific PCR and gel electrophoresis. As our hypothesis stated, no band is visible using the mutant primer, and our wild-type primer shows minimal binding at 392 base pairs when compared to the ladder (Maison, 2009).

To fully understand the scope of cystic fibrosis and other genetic diseases, surveys and interviews were conducted with professionals in this field. Stephanie DeWard MS, CGC, a genetic counselor, and Wendy Bouck LRT/RRT-NPS, a respiratory therapist were asked multiple questions regarding the psychological and sociological impact of cystic fibrosis. Answers were given on a numerical scale as well as open-ended informational answers.

Results of PCR and Informative Survey*

Our group initially predicted that the design of two primers, one mutant and one wild-type, would yield one band using allele specific PCR. The correct PCR cocktail and annealing temperature would be needed to obtain this band. This prediction was founded on the basis of the difference in primers. The mutant primer was constructed to bind only to mutant DNA, which contained the mutation 1078 del T. The deletion of Thymine causes a frame shift, and would not allow the mutant primer to bind to wild-type sequence. In contrast, the wild-type primer was constructed without the mutation, and the shift, allowing it to bind only to wild-type DNA. This successful binding and amplification was expected to be at 392 base pairs using gel electrophoresis. It was also hypothesized that results from the survey would show a bigger burden is placed on familes with CF(Blair et al 1995).

Many PCR were conducted before obtaining bands. After the first two failed attempts, our group recognized our PCR cocktail was incorrect. Only 1mL of 1mM dNTPs was used. This concentration was then corrected to be 10mL of 1mM dNTPs., This altercation caused the concentration of water to be reduced from 40mL to 31mL. This change was needed to keep the amount in the PCR cocktail below 50mL. The improved cocktail was used throughout the duration of the experiment. Three more failed PCR were conducted using the correct cocktail before discovering the flaw in our reverse primer. While constructing the reverse primer, it was designed from the 3’ to 5’ end. When ordered, the primers needed to be oriented in a 5’ to 3’ direction, and the sequence was not reversed during this stage. Once this mistake was realized, reverse primers were re-ordered, and were then used throughout the experiment. Annealing temperatures were varied with each trial.

Although the original cocktail, and reverse primer was incorrect, our group concluded that new DNA should be purified. The original DNA purified was that of an IB3 bronchial epithelial cell. The re-purified DNA was that of an S9 cell is an airway epithelial cell, which expresses wild-type CFTR and is derived from IB3 cells (Schneider et al 1999). The concentration of the IB3 and S9 DNA using mass spectrometry was 14.78 ng/mL and 60.759 ng/mL respectively, and S9 cells were used for the rest of experimentation.

Using the new DNA, reverse primer, and cocktail allowed us to focus on the annealing temperature. Four PCR were completed with the above corrections. Once all mistakes were corrected, we returned to our predicted annealing temperature of 56°C as calculated by Tm= 100.5 + (41 * (yG+zC)/(wA+xT+yG+zC)) - (820/(wA+xT+yG+zC)) + 16.6*log₁₀([Na⁺]) where w, x, y, and z are the number of A, T, G, and C in the primer (Kibbe, 2007). Starting at 54°C and 52°C, the annealing temperature was adjusted according to the obtained results. No bands resulted in annealing temperatures from 45°C-56°C. This wide range of attempted annealing temperatures indicates the equation used was not the correct one to use, and may not be useful for primers of our length or composition. On the fourth and final PCR, annealing temperatures of 40°C and 42° C were chosen. Both of these temperatures resulted in non-specific binding of our wild-type primers, and no bands for the mutant primer.

Although the results were not definitive, bands of indiscriminate binding show correct primers were used, as there is binding at the predicted band length. To make the band more viable, the annealing temperature could be raised until a clear band is present. Also the cocktail may need to be adjusted to provide the best results. Previously, scientists were able to create primers that could detect our mutation, however, they used a different method called PCR-mediated site-directed mutagenesis (PSM). This involves using restriction enzymes and a single base mismatch at the mutation site (Dorval et al 1994). If the mutation does not affect the restriction enzyme site, it PSM will make a single base alteration. This results in constraint, and results in 2 bands in the wild-type sequence, and 3 in the mutant (Dorval et al 1994).

Our survey indicated a positive correlation of family strain with cystic fibrosis patients. Both DeWard and Bouck agree that sibling jealousy is a major concern. Bouck states, “There is definiate sibling resentment, but it goes both ways. The sibling of the patient may feel neglected because of the attention given during treatments, but the patient may act out because they are not as able to be ‘normal’”. Both again agree that increased public knowledge has increased demands for research on genetic diseases. DeWard and Bouck answered the question “Has increased public knowledge of genetic diseases cause and increased demand for medical research?” on a numerical scale, 1 being little and 5 being much. DeWard answered 4 and Bouck answered 5. As with many other diseases, there are influential celebrities and organizations that help fund and put these in the public eye. Former pro-football quarterback Boomer Esiason has a son with cystic fibrosis, and has a college scholarship in his name for patients affected with cystic fibrosis. All of these factors help in changing public opinion on the disease. DeWard explains, “The more the public is aware and talking about these diseases, the better it is for everyone”. However, Bouck and DeWard disagree on how public opinion may affect someone with cystic fibrosis. Both answered the question on the same numerical scale. This time DeWard said 1 while Bouck said 4. Deward said, “Patients with CF do not care what others think. They are their own person and the mentality that I can do anything I set my mind to”. Although Bouck agrees some have this gift, others find it hard to get married or hold a job.

Future Directions

The experiment could have been improved with more time and research. Initially, the reverse primer was ineffective. After several weeks a new reverse primer was used, which annealed, resulting in non-specific binding. Some of the trials (using the ineffective reverse primer) were deemed inconclusive because of an incorrect dNTP concentration. With further research and more time, we feel our assay would have been diagnostic.

In order to ensure our test was diagnostic, more controls should be used. The DNA from the IB3 cells contained a CF mutation. Probability (based on frequency of CF mutations) suggests that the mutation was ∆F508 (Schneider et al., 1999). Additional testing (including primer design and an additional diagnostic assay) should have been undertaken to confirm that the mutation present in the IB3 DNA was ∆F508. A suggested control for future research is the use of restriction enzymes. Because restriction enzymes cleave DNA strands at code-specific regions, they can be used to break apart a strand of DNA and lend support to its identity. This way, two bands of the same length can be differentiated based on composition.

Also, different methods of allele-specific PCR and primer design could be used. Rapid Cycling PCR can increase efficiency and save time and money compared the type of PCR used in this experiment (Wittwer et al., 2009). Also the Yaku-Bonczyk method could be used for primer design of the ∆F508 mutation (Cracolici, 2009).

Figures

Written By: A40703106

Revised by: A40525550

Finalized By: A40172438

1 2 3 4 5

Figure 5. Amplified S9 DNA containing CFTR mutation 1078delT using lower annealing temperatures. Thermocycling was ran for 30 cycles at 95°C for 5 minutes in the first cycle then 95°C for 30 seconds, 40°C or 42°C for 30 seconds, 72°C for 45 seconds, and during the last cycle 72°C for 5 minutes. The cocktail contained 31 μL of water, 1 μL of forward primer, 1 μL of reverse primer, 10 μL of 1X dNTP, 1 μL of IB3 DNA template, 1 μL of Taq polymerase and 5 μL of PCR buffer. Lane 1 contains the 1KB ladder. Lanes 2 and 3 contain PCR product with mutant and wild type primers respectively at an annealing temperature of 42°C. Lanes 4 and 6 contain PCR product with mutant and wild type primers respectively at an annealing temperature of 40°C. Lane 5 is empty due to error. Lane 3 contains non-specific binding meaning the annealing temperature is too low. The bottom band of the non-specific binding is located at around 392 base pairs.