The recessive genetic disease of Cystic Fibrosis results from the improper coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel in epithelial cells. G85E is one such mutation that leads to CF and has a worldwide prevalence of 0.2%, most common with those of Mediterranean descent (Decaestecker et al., 2004).
Between the primers developed via the traditional and Yaku methods, it was hypothesized that the primers developed from the Yaku method would more consistently and accurately binds to the template DNA with the G85E mutation present than primers developed using the traditional method. The Yaku primers are still able to bind to the template DNA, even with the third base pair being a mismatch, as long as the following two base pairs match the template DNA and anneal. The strength of the following base pair bonds after the mismatch is enough to overcome the mismatch, thus creating a stronger primer, as the primer would not anneal with only the strength of one subsequent match. Both types of primer creation methods are proven to result in smearing at low annealing temperatures (beginning at 45°C) and show no discrimination against non-specific binding (Figure 1 and Figure 2). Although the traditional method is effective the majority of the time, the Yaku method has proven to be the superior technique for primer creation becoming more evident at higher annealing temperatures (47°C), which can be seen by the slightly smeared bands in lane 6 and light banding in lane 12 with primers of the Yaku-method as compared to less distinct band(s) in lane 3 and the somewhat smeared false positive band in lane 9 with primers of the traditional method (Figure 3). Upon an increase of the temperature to 49°C, the traditional method primers continue to result in mild non-specific binding in lane 4 as well as a false positive band at 600 base pairs in lane 10, while the Yaku method primers resulted in a single distinct band at 600 base pairs in lane 7 and no false positive band in lane 13 (Figure 4) (Wu et al., 2009). Although trials with annealing temperatures up to 52°C were run, there was no change in band distinction between traditional and Yaku methods at the higher temperatures, nor did the mutant traditional method primer cease to result in a false positive band, indicating that the Yaku method was superior for this assay.
As the G85E mutation causes an uncomplicated but critical change on the coding of the CFTR gene, it is the simplicity of this change that makes the Yaku method more favorable as well. With only one base pair alteration on amino acid 85, guanine shifting to adenine, as opposed to an amino acid deletion such as in the DF508 mutation, the Yaku method is more effective because it specifically targets the altered base pair (Decaestecker et al., 2004; Yaku et al., 2008). However, it may also be argued that because of this same simplicity of the mutation that the traditional method of primer creation is equally as effective. The traditional method takes into account the change at the 368th nucleotide and is also more applicable to mutations such as the DF508 mutation; still, the Yaku methods’ reliance on the strength of nucleotide bonding will usually result in more consistent and clear readings on gel electrophoresis tests as seen from the tests on the G85E mutation.
Essentially only one protocol was used in the experiment, although several trials were done using different annealing temperatures to yield more credible bands. Multiple variations in annealing temperature were focused upon to discern the consistency of the primers. Effects from variations in other aspects of the protocol such as salt concentration or cycle times and number, however, were not ascertained because ultimate distinction of bands at 600 base pairs was not the purpose. Still, though bands were produced that support the hypothesis, this lack of variation undercuts the validity of the conclusions made. Although, based on other sources, the results produced from these tests may hint at the conclusion that the Yaku method is superior to the traditional method for primer creation, it will never be statistically significant in this case because the experiment has not been through multiple varying trials (Yaku et al., 2008).
Further research can be done to explore the Yaku method of making primers. Alterations could be made to the primer to increase the chance of accurate binding to the template DNA. In this case, the single intentional base change was to a cytosine from a thymine, which increased the guanine/cytosine content, thus resulting in a higher and thus more desirable annealing temperature. If further alterations do increase the accuracy and consistency of primers created through the Yaku method, it would also increase the ability to add to the guanine/cytosine count (which enables the annealing temperature to rise). Similarly, varying placement of the intentional mismatch could yield a more discriminating primer, resulting in fewer false positives. Although the effect of salt concentration is known to produce a more distinct band (less non-specific binding), perhaps the concentrations may effect different versions of the Yaku method in varying positive or negative manners. Tests run to discern these effects could also yield better methods of diagnostic assay creation (Haenisch, et al., 2009).
The social experiment on disease allowed an assessment of how the public views diseases and the difference in opinions based on whether the disease is contractible or not. It was hypothesized that the public would be more accepting towards a display of a known genetic disease, such as Cystic Fibrosis, than they would be towards a display of the “Gay Gene”. Variation in public awareness and media representation highly influences the reactions of the public (Conrad and Markens, 2001). In a previous study, negative responses were seen through a study done on homosexual discrimination where 37% of homosexual men had anti-gay verbal abuse within a six-month period (Huebner et al., 2004). Through a display at the corner of Farm Lane and Auditorium Road, a sample size of 50 people per display was gauged for responses of hostility (anger) or unknown (ignoring the sign, showing interest or confusion). The resulting number (8) of hostile responses to the “Gay Gene” display appeared significantly different than the number (0) of those to the “CF Gene” sign (Figure 5 and Figure 6). However, these results were not statistically significant, having a resulting p-value of 1.00 (Table 1). In terms of statistical significance, the hypothesis was false, nevertheless, is remains important that the “Gay Gene” display evoked hostility where the “CF Gene” display did not.