Creation of a Diagnostic Assay to Detect R553X Mutation in CFTR gene in Human DNA using PCR

By: B998, B898, B644, and B407

LB 145 Cell and Molecular Biology II

Tuesday and Thursday 12:40pm-2:30pm

Andrew Vanalst, Davin Hami, Emily Nemeth

April 23, 2019

Title Page Authored By: B407

Title Page Revised By: B644

Title Page Finalized by: B898

Consent has been given to participate in the Extra Credit 30 days Experiment

Abstract

Authored by: B407

Revised by: B644

Finalized by: B898

The R553X mutation is one of more than 1,700 mutations of the CFTR gene that are known to cause the autosomal recessive disorder, Cystic Fibrosis (CF) (Bal et al. 1991). This mutation is caused by a cytosine to thymine substitution at base 1789 in exon 11 of the CFTR gene. This causes a change in code of the amino acid from arginine to a stop codon. (Hull et. al., 1994). Unfortunately, detecting this mutation has been expensive and invasive for patients. The purpose of this experiment was to design a diagnostic assay that eliminated the invasive factor by using polymerase chain reaction (PCR) and experimentally designed primers to detect the R553X mutation in patients with cystic fibrosis. Our designed primers specific to the R553X mutation will bind to the nucleotides on the 11th CFTR exon of mutant or wild type DNA. We hypothesize that the intentional mismatch at the 3’ end of our primers (mutant type or wild type) will create steric hindrance during binding, reducing non specific annealing to more efficiently amplify the target sequence  in patients who may have the R553X mutation (Yaku et. al, 2014). Once our DNA sample underwent PCR, it was analyzed using gel electrophoresis. By observing the gel under UV light and using a DNA ladder, we expect to produce a band of 1010 base pairs. A band at this length will confirm or deny whether or not the DNA sample contained the R553X mutation. Depending on whether or not the DNA is mutant type or wild type, there are 2 instances in which bands could be produced. A band that appears in the presence of our wild type primer will suggest that the DNA is absent of the R553X mutation. Whereas, a band produced in the presence of the mutant type primer suggests that the DNA contained the R553X mutation. Through the use of 2 distinct primers, two bands can be produced (Hamosh et. al., 1991). We predict that designed primers would detect the R553X because PCR is an effective method of amplifying target DNA to view genetic mutations (Felix et, al., 2001).

Figure 8. Amplification of human buccal cells, using published primers. The figure above is a representation of amplified human genomic DNA with published primers. Fluorescence of DNA is shown with the use of SYBR safe dye in a 1% agarose gel. The gel consisted of 38 ml of deionized water, 2 ml of 20X lithium borate, and .4 grams of agarose powder. After combined, the ingredients were gently mixed and microwaved for 15 seconds. The mixture was then gently swirled and microwaved for 15 more seconds. The gel mixture was then cooled for 5 minutes and 4µl of SYBR safe dye was added. The gel was ran at 200 V for approximately 29 minutes, the duration of time for the dye to run 75% of the way down the gel. Published primers from a prior research paper were used in this experiment to amplify wild type DNA from human buccal cells. The primers were as follows, The published reverse primers, obtained from Syd Labs.  These primers were expected to amplify the DNA at approximately 291 base pairs. Using a semi-log plot, it is shown that DNA was amplified at 288, 306 and 308 base pairs respectively. In the equation displayed on the graph, the y represents the number of base pairs detected, whereas the x value represents the number of pixels that the band travelled. In lane 3 PCR41A was pipetted in the well. All three PCR cocktails had a DNA concentration of 7.1ug/ul. PCR41A and PCR41B contained 70ul of DI H2O, 10ul of 10x buffer, 2µl of DNTPs 3ul of both forward and reverse primers, and 3µl of TAQ polymerase and MgCl2. The difference between A and B is the volumes pipetted into the wells. A had 8ul of cocktail and 2µl of bromophenol blue dye. In wells 7 and 9, PCR42A and PCR42B are present, respectively. For the contents of this cocktail, it was 68ul of H20, 14µl of 10X buffer, 2µl of DNTPs, 3µl of both forward and reverse primer, 2µl of DNA, 3ul of TAQ polymerase and 3µl of MgCl2. We predict that this was unsuccessful because of the ratio of buffer to DNA. There should have been a larger amount of buffer for the DNA to be amplified, if there was then this could have been a successful cocktail. Finally, in wells 11 and 13, PCR43A and B are present. For the contents of this cocktail, it was 60ul of H20, 20µl of 10X buffer, 2µl of DNTPs, 3µl of both forward and reverse primer, 2µl of DNA, 3ul of TAQ polymerase and 3µl of MgCl2. Notice the streaking in wells 3,5,11 and 13. This is due to a possible fragmentation of DNA, which causes smaller and larger fragments of DNA to move farther or not far enough in the wells because of their different sizes.

Discussion

Authored by B-644

Revised by B-407

Finalized by B-998

Experiment Summary

        Cystic Fibrosis is a genetic disease found commonly in caucasians throughout Europe and North America (O’Sullivan et al., 2009).  Cystic Fibrosis is a recessive disorder caused by mutation in the gene which codes for the cystic fibrosis transmembrane conductance regulator (CFTR), which is a membrane protein that acts as a chloride channel. This protein pumps chloride ions across the epithelial cell membrane (Gadsby et al., 2006). The CFTR protein is responsible for transporting these chloride ions across the cell membrane which help regulate salt concentrations throughout the body, mostly affecting the respiratory and digestive system, as well as the reproductive system (Gadsby et al., 2006). Unfortunately, a mutation of this gene causes a misfolding or loss of function of this protein, therefore causing an ion exchange imbalance (Welsh et al., 2001). This imbalance can result in dehydration of the mucus in the excretory organs, most severely in the respiratory tract. An unbalance of these chloride levels could also affect the pancreas, gastrointestinal tract, sweat glands, and other exocrine tissues (Knowles et al., 2002).

The R553X mutation is known as a nonsense or stop mutation on exon 11, which results in the skipping of this exon (Hull et al., 1997). A nonsense mutation occurs due to a premature stop signal that causes the production of the CFTR protein to cease prematurely. In this case, the cell believes that is has reached the end of the instructions for this code and ceases production of the protein that is necessary to function (Walkowiak et al., 2005). Therefore, in this class one mutation, there is no functional CFTR protein that is produced at all. This can cause severe and lethal health issues if not properly treated, documents of pancreatic failure have been studied and compared to the other classes of mutations and it has been found that class one and class two mutations in patients have lead to higher amounts of insufficiency of the pancreas (Walkowiak et al., 2005). Treatment options are limited, but detection of this disease is most important. During this study of the R553X mutation, we hypothesize that by designing both wild-type annealing and mutant annealing primers to use in the process of PCR that have an intentional mismatch 3 base pairs away from the 3’ end to increase the likelihood of steric hindrance and reduce non-specific annealing that we will more efficiently amplify targeted sequences of DNA. Our thesis is that by creating a successful diagnostic assay with the use of our designed primers and PCR through gel electrophoresis, the R553X DNA mutation target sequence will be more highly and efficiently amplified, and therefore increase the likelihood of being correctly identified, all in an effort to lead to advances in the medical field as it will be a simpler and less expensive means of identifying this mutation in CF patients (Yaku et al., 2008)

Original Predictions

 As of 2008, the sweat chloride test remains the standard for diagnosing cystic fibrosis, however, it does not always provide a clear answer (Farrell et al., 2008). The purpose of this study is to determine a diagnostic assay to correctly identify a specific mutation of cystic fibrosis called R553X by developing a new PCR technique.  This specific mutation replaces the arginine amino acid with a stop codon and this stop codon will prematurally stop the CFTR protein when being assembled which results in a product that will not work, as stated above (Bal et al., 1991). Before use of our designed primers, we created an assay involving a set of published primers to test on human genomic DNA. These published primers will bind to the target strand of DNA and, once ran through gel electrophoresis, we predict will amplify a band length of 291 base pairs. This expectation depends on the involvement of the common forward primer, reverse wild-type primer, and reverse mutant type primers because of the deletion of exon 11 found at the base pair placed at the end of the primer (Lago et al., 2017). During research for this experiment, a new set of primers were designed that are more effective for targeting point mutations by using the Yaku-Bonczyk method to place an intentional base pair mismatch 3 bases from the 3’ end of the primer (Yaku et al., 2008). We predict also that after running gel electrophoresis for the mutant type and wild-type DNA there would be a distinct band at 1010 base pairs in length because both the mutant-seeking and wild-type-seeking primers are designed as allele specific to only bind to the DNA they are designed for (Ng et al., 1991). 

Results and Findings

After calculating the annealing temperature, it was found to be 51℃, but after conducting control experiments, it was found that 50℃ would be a more optimum temperature because it was not too hot, which would denature the DNA, and it is better to be slightly below the temperature calculated to account for error in temperature management (Fernández-Lorenzo et al., 2009). After running PCR, gel electrophoresis was run and assessed under a UV light to assess whether or not a band was detected. When it was placed under this UV light, we could recognize whether or not there was DNA separation occurring. During our positive control, using lambda virus genomic DNA, PCR was conducted to understand the thermocycler and to amplify this DNA with an expected band length of 394 base pairs. During this experiment, we produced a PCR cocktail and ran it through the gel, and therefore we were able to produce the ladder (KB+), but were unable to produce a band (Trial 2). We predict this failure to produce bands may be due to an absence of Mg+2 into the PCR cocktail because it is essential to help the enzyme TAQ polymerase function (Hull et al., 1994).  We made note of this error in our positive control and continued on in the experiments that followed making the PCR cocktails with Mg+2, therefore allowing the TAQ polymerase to function properly. We were then able to formulate a successful lambda virus PCR detection assay in which a band of 394 bp was evident compared to the Kb+ ladder.

Upon determining the efficiency of the PCR process itself, we were then able to further our research in obtaining wild-type human genomic DNA via a Chelex resin protocol. We were unable to detect the presence of any DNA through PCR initially and predicted that we had not adequately collected enough DNA because of a lack of centrifugation to more effectively separate the cheek cell debris from the DNA contained in the supernatant. We also predicted that the ratio of Taq to DNA was inadequate for the concentrations we had determined (Huang et al., 1992). Ultimately, we adjusted the ratio of Taq to DNA by increasing the quantity of Taq, as well as increasing our centrifugation times and temperatures to more effectively separate the DNA-containing supernatant from the pellet of cheek cells that remained after centrifugation. We were then able to run this newly obtained DNA through PCR where we achieved gel electrophoresis products that amplified 288bp, 306bp, and 308bp bands. This result further supported our hypothesis in use of the published primers to amplify a region of DNA located on the CFTR gene(Lago et al., 2017).

Again, when running the human genomic DNA PCR cocktail with our designed mutant seeking and wild type seeking primers, we predict that there will be a band showing on the gel at 1010 base pairs in length because both of these designed primers are allele specific to bind to the DNA that they are designed for (Ng et al., 1991).

These designed primers will be more effective at diagnosing patients with the R553X mutation, and thus can help diagnose patients earlier in their lives. Earlier screening and detection for cystic fibrosis can lead to specialized medical care and therefore, can improve future outcomes. Apart from improved health of people who are treated earlier on, this can also cut down on medical expenditures by those who are afflicted with it. In 2006, the average annual cost of a patient with cystic fibrosis was $48,098 which is 22 times higher than the average person's medical expenses (Ouyang et al., 2009). Cystic fibrosis can also cause organ failure in some parts of the body which requires transplants, yet again increasing the patient's annual expense (Ouyang et al., 2009). If cystic fibrosis is detected earlier on, then better medical care can be available which can help reduce the chance of organ failure later in life and could help save the patient from extensive medical expenses. As you can see, earlier detection can not only improve a person’s health, but could also help cut back on these costs. After conducting this experiment, it is evident that research similar to this PCR testing, future studies that concern R553X and other mutations of the CFTR protein may be assisted in the generation of extensive clinical diagnostic support.