Greater Specificity in Yaku versus Traditional Primer Design in AS-PCR Diagnostic of G85E CFTR mutation in IB3 C

Team Lao Hu
Jillian Braid, Natalie Palumbo, and Christine Plichta

Abstract

 

The G85E mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) occurs in less than about 0.2% of Cystic Fibrosis (CF) patients (Decaestecker et al., 2004). The protein alteration is the result of a substitution of adenine for guanine on the third exon at nucleotide 368 of mRNA (Decaestecker et al., 2004). The G85E mutation is a class II, missense mutation that alters glycine at amino acid residue 85 to glutamic acid (Kerem et al., 1997).  Using polymerase chain reactions (PCR) and subsequently gel electrophoresis, the DNA extracted from human IB3 bronchial epithelial cells of CF patients was analyzed during the diagnostic assay. Two methods of primer creation were used to generate four primers, two wild-type and two with G85E mutated, via the traditional method and the Yaku method, the latter which involves the single mismatch of a base pair two nucleotides away from the finish of the 3’ end. This was done to test for the most successful diagnostic assay for the mutation’s presence. We hypothesized that of the four primers created, the primers formulated using the Yaku method will be more accurate and consistent in detection of the G85E mutation because the mismatched base pairs have greater effect on annealing, as a bond from only one base pair match between the two mismatches is not strong enough to allow for extension (Yaku et al., 2008). Annealing temperature started at 45C and at 49C resulted in defined bands at 600 base pairs of both wild-type primers and the traditional mutant primer. While traditional method primers showed some non-specific binding, wild-type Yaku primers resulted in a brighter band and mutant Yaku primers resulted in no band, as predicted. These results may lead to a shift in the conventionally used method of primer design for fewer false- positives. In relation to the study of CF as a genetic disease, a comparison study of the attitudes of the Michigan State University student population was conducted between a genetic disease (CF) and a controversial genetic issue (the Gay Gene). Signs displaying “Gay Gene?” and “Cystic Fibrosis Gene?” were individually displayed for one hour in a moderately trafficked area and passerby’s reactions were recorded. We predicted more hostility toward the Gay Gene, as there is debate about its existence. However, the difference between numbers of hostile reactions was not statistically significant (resulting p-value of 1.00). This indicates that idea of homosexuality as a result of genetics, similar to cystic fibrosis, may be more accepted than previously thought.