BUNCH of ASPARAGUS

Amplification of Cystic Fibrosis W1282X Mutation in

HeLa cells using Yaku Primer Design yields Ambiguous Results

Jacob Aubry, Jillian Harold, Emily Schmitt-Matzen, Soo Hur

LB 145 Cell and Molecular Biology

Tuesday and Thursday 12:40 PM

Dr. Douglas Luckie, Katie Oleski, and Natasha Bhaskaran

4/15/11

Abstract

The Cystic Fibrosis (CF) W1282X mutation results from a single nucleotide substitution in exon 20 of the CFTR gene (Feriotto et al., 2001). Tryptophan is replaced with a stop codon when the first Cytosine in the codon ACC is replaced with Thymine (Shoshani et al., 1992). The purpose of this study was to successfully create an assay detecting the W1282X mutation using PCR and gel electrophoresis. It was hypothesized, primers designed with an intentional mismatch on the third base pair from the 3’ primer end, according to the Yaku-Bonczyk method, would not provide enough binding strength for primers to anneal to non-complementary sequences, reducing non-specific binding; along with specified PCR thermocycling and gel electrophoresis this would unambiguously determine the presence of the W1282X mutation gene sequence (Yaku et al., 2008). It was predicted that Fprimer1 present with wild-type DNA would result in a band of 1618bp, and a 1619bp band would result when Fprimer2 and mutant DNA were present (Feriotto et al., 2001). Positive control primers were also designed with an 839bp band expected. The results were inconclusive as non-specific binding occurred regularly. A sociological study was completed to assess the public’s social responses to the CF symptom of persistent coughing. Based on a survey, it was predicted 71.7% of people present in four public locations would have negative responses to an individual persistently coughing. A chi-squared test was completed resulting in a P-value of 0.9, suggesting the observed and expected percentages were statistically significant. Overall, this assay was created to test the Yaku primer design and its usefulness in the diagnosis of the W1282X mutation.

Discussion

Experiment Summary

Cystic Fibrosis is the most common autosomal recessive genetic disease in Caucasians (Moss, 1995). It often arises due to improper encoding for a protein caused by a DNA sequence mutation, resulting in dysfunctional chloride channels in epithelial cells (Wilschanski et al., 2003). The W1282X mutation is a specific point mutation prevalent among Ashkenazi Jewish community (Shoshani et al., 1992). This mutation occurs when a cytosine is replaced by a thymine; consequently a stop codon is encoded for instead of tryptophan (Shoshani et al., 1992).

Overall, it was hypothesized that primers developed using the Yaku-Bonczyk method would reduce non-specific binding because the intentional mismatch three base pairs from the 3’ primer end would decrease the tendency for the mutant and wild-type similar primer sequence to anneal to uncomplimentary DNA sequences (Yaku et al, 2008). According to the Yaku method, if the primer attempted annealing to the correct DNA sequence the strength of subsequent base pair bonds after the mismatch overcomes the mismatch and continues extension, resulting in a more specific binding (Yaku et al., 2008).

Original Predictions:

The presence of W1282X mutation in the PCR amplified HeLa DNA products was determined by using gel electrophoresis (Shoshani et al., 1992). In the research, two forward primers: Fprimer1 and Fprimer2 were designed using the Yaku method to distinguish between the wild-type and mutant CFTR gene based on a single intentional base pair mismatch. Also, a reverse primer was created to amplify target DNA with either Fprimer1 or Fprimer2 to correctly diagnose the presence or absence of the W1282X mutation in CFTR gene (Feriotto et al., 2001). Moreover, positive control primers were developed to examine whether the PCR cocktail and thermocycling worked properly (Figure 1). Expected band sizes were determined to be at 1618 base pair in homozygous wild-type genotype with Fprimer1 set, while homozygous mutant genotype with Fprimer2 set was 1619 base pairs. Heterozygous individuals, with Fprimer1 and Fprimer2 were expected to show faint bands at 1618 and 1619 base pairs due to the having both a mutant and wild type allele (Feriotto et al., 2001). The control primers were expected to have bands at 839 base pairs.

Ultimate Findings:

The Purpose of this study was to create a PCR assay using the designed primers developed using the Yaku-Bonczyk method that would unambiguously determine the presence or absence of the W1282X mutation in human HeLa DNA. In an attempt to create this assay, DNA concentration and annealing temperatures, which could directly affect the annealing rates and specific binding of designed primers, were altered (Shoshani et al., 1992). The DNA concentrations needed to be increased and at least 15 μl of PCR product needed to be loaded into the gel for bands to be detected.

After this adjustment, both Fprimer1 and Fprimer2 resulted in no specific bindings at a low 49°C annealing temperature while non-specific bindings were observed at 50°C (Figure 2). Additionally, the control band was not located at the expected band length of 839bp, however, it was a distinct band and the ladders were not extremely precise enough to determine the band size (Figure 2). Furthermore, the control primers produced a band consistently in the same location.

When the annealing temperatures were raised to 49.5°C and 50°C, non-specific binding was seen (Figure 3a). To troubleshoot, the annealing temperature was increased to create more specific conditions for annealing. Ideally, the increased temperature would increase the difficultly of primers to anneal to the non-complementary strand, thereby reducing non-specific binding, yet be low enough that binding to the complementary strand could occur often (Yaku et al., 2008). When the annealing temperature was raised to 50.5°C, there was less non-specific binding, yet there was a band present below the 100bp mark on the ladder (Figure 3b). This indicates that the primers were accurately annealing to the DNA template. However, the band does not appear to be a primer dimer because there is no indication of a distinct lower band in annealing temperatures below 50.5°C (Figure 3b). For the control primers, optimal annealing temperature was determined to be at 49°C-50°C because non-specific bindings became more evident upon increasing the annealing temperatures to 50.5°C, 52°C, and 53°C (Figure 3a, 3b and 4).

No distinct bands could be seen for temperatures above 50°C in other trials which suggests that the best annealing temperature for theFprimer1 and Fprimer2 developed via Yaku method are 50°C. However, due to the absence of distinct bands for any of the amplified PCR products with varying annealing temperature, the hypothesis that these specific primers would reduce non-specific binding, leading to a less ambiguous determination of the W1282X mutation’s presence was not supported.

If a different primer design method was used, better results may have occurred. This is a possibility because although there are significant amounts of data and research that support the Yaku method, there are circumstances and technical difficulties that could inhibit the success of the assay such as the specific point in the W1282X mutation and its surrounding base pairs (Yaku et al., 2009).

Sociological Experiment

The sociological experiment on CF assessed public perceptions of CF symptoms. People’s responses were recorded in four difference locations: an elevator, cafeteria, study lounge and restaurant over 30 days. It has been determined that about 31% of the CF patients aged 16-18 feel emotionally disturbed when displaying CF symptoms in public (Cowen et al., 1984). However, few studies have been performed regarding the public’s reactions to CF symptoms. To examine this sociological issue, a brief online survey was conducted to determine the percentage of negative reactions among college students aged 18-22 around individuals exhibiting one of the most common CF symptoms such as persistent coughing. According to the survey data, 71.7% of 105 people felt uncomfortable around people displaying the symothoms associated with CF, such as persistent coughing. Moreover, 52.66% would opt to take the stairs if they witnessed someone coughing on an elevator. Lastly, only 32.7% of people considered that persistent coughing may be attributed to a genetic disease (Figure 5). Based on the survey, it was hypothesized that 71.7% of observed individuals were expected to show a negative reaction around CF symptoms in various public locations. Numerous adverse reactions such as staring and moving farther away from individual displaying CF symptoms were observed. Overall, the P-value of a chi-squared test was determined to be 0.90, which is greater than 0.05, thus the hypothesis that 71.7% of people feel uncomfortable around CF symptoms is accepted which indicates there is a 90% probability that deviation from the hypothesis is due to chance alone (Table 1). Ultimately, the sociological experiment supported the data revealing that 31% of CF patients experience emotional disturbance and feeling judged based on their CF symptoms in public because over the 70% people expressed negative reactions towards individuals with CF like symptoms.

Future Experimentation

The Yaku primer design did not yield unambiguous results for the W1282X mutation in this specific assay. Less binding was seen in the mutant primer and this may have been a result of the A/T matches after the intentional mismatch. Since an A/T match only has two hydrogen bonds, instead of three that are present in a G/C match, less amplified product is usually seen with an A/T match (Yaku et al., 2008). This combined with the need for the A/T matches to overcome the mismatch may have resulted in not as much target DNA amplification, resulting in less distinct bands.

Further research can be done to advance the diagnostic ability of allele specific PCR for cystic fibrosis, specifically the W1282X mutation. Additional research could be done comparing traditional primer design methods to the Yaku primer design method. The traditional primer for the wild-type DNA sequence would be 5’-AT CTT CTT CAT TGC TGG AC-3’, binding to the anti-sense strand. The traditional primer for the mutant DNA sequence would be

5’-CAT CTT CTT CAT TGC TGG AT-3’, binding to the sense strand. A higher G/C concentration in the mutant primer on the third base pair from the 3’ end might help binding and enhance the chance of primers to efficiently anneal to the DNA template thus, reducing the non-specific binding (Nir et al., 2002). Also, in the traditional primer method there is no need for the base pairs after the mismatch to overcome the mismatch. Alternation of primer using the traditional method will may improve annealing because there is no intentional mismatch (Feriotto et al., 2001).

By taking various approaches, science can progress and gain more knowledge on the finest approach to diagnose CF and other genetic recessive diseases. Assays such as ours can be implemented into testing for allele frequencies of the W1282X mutation in different ethnicities, and then these can be compared to the allele frequencies between the same race in different regions of the world. Essentially, there are many studies that can be done using our approach; and, as a result, the success of these future studies helps further verify our assay through its implementation and usage in these future studies.

Figure 2. Gel Electrophoresis of PCR products from CF HeLa cells containing the W1282X mutation for annealing temperatures of 49°C and 50°C. L1 of a 1.0% LB agarose gel was loaded with 2µL of Kb Plus ladder with 3µL bromophenol blue loading buffer dye. The remaining wells were loaded with PCR products containing 6ng of DNA in 15µL of PCR product which had been previously mixed with 3µL of bromophenol blue loading dye. L2, L3, and L4 had an annealing temperature of 49°C and L5, L6, and L7 had an annealing temperature of 50°C. L2 and L5 were loaded with PCR products of the wild-type primer, Fprimer1, and the reverse primer with an expected band length of 1618bp. L3 and L6 were loaded with the mutant primer, Fprimer2, and reverse primer PCR products with an expected band length of 1619bp. L4 and L7 were loaded with the forward and reverse control primer PCR products with an expected band length of 839bp. Each lane was loaded with 15µL of PCR product which had been previously mixed with 3µL bromophenol blue dye. L5 and L6 indicate non-specific binding due to DNA fragments evident from approximately 5,000bp to 500bp. L5 has brighter non-specific binding with a more distinct band at approximately 1,620bp which is near the expected band length of 1618bp for wild-type DNA with Fprimer1 and Rprimer. L4 and L7 have distinct bands at approximately 1650bp indicating a reproducible band for the control primers.