The G542X mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the second most common allele found in cystic fibrosis (CF) patients in the United States, accounting for 4.6% of all CF cases. This nonsense mutation consists of a single nucleotide change from G to T, altering glycine (GGA) into a premature stop codon (TGA) at the 542^nd amino acid on exon 11 of the CFTR gene. The purpose of this study is to create a more robust allele-specific polymerase chain reaction (PCR) assay to diagnose the G542X mutation. We hypothesized that, through the Yaku method, the incorporation of additional intentional nucleotide mismatches at the 3’ end of primers would reduce false positives in PCR due to increased primer discrimination. Positive controls were regularly run to establish reliable PCR protocols and template integrity. Amplification of Lambda virus Rz gene using Rz1F and Rz1R primers was utilized as a ‘PCR control’ that produced a 395 bp product when reagents were optimal and PCR of purified template with published primers versus CFTR was conducted as a CFTR ‘template control’ which produced a product size of 256 bp. In developing our novel AS-PCR assay we designed PCR primers using the Yaku group’s intentional mismatch strategy. These ‘Yaku primers’ were designed to amplify an 855-nucleotide region to diagnose the presence of either mutant G542X-CFTR or wild-type CFTR in samples. Amplification of mutant and wild-type CFTR DNA samples was performed producing an appropriate ~859 bp length product supporting that the Yaku primers were successful in G542X detection. Furthermore, Yaku primers designed to identify wild-type CFTR did not also amplify a similar product from samples containing mutant G542X-CFTR template, supportive that the primers limited false positive results.