Cystic fibrosis transmembrane conductance mutation detection using fluorescent hybridization probes and melting curves

Abstract

Background/aims Single-point mutations or single-nucleotide polymorphisms, deletions, and insertions in genetic sciences are related to several human diseases, such as cancer, metabolic disorders, some types of mental illness, cardiovascular diseases, diabetes, etc. Consequently, precise, fast, and sensitive detection of these mutations in specific genes has substantial value in disease diagnosis, in the forecast of patients’ responses to treatments, threat of deterioration of diseases, and outcomes. However, the existence of minute differences in structural and conformation dynamic stability from single base or multibase mismatches between the wild type (WT) and its mutated targets makes detection convenient. The common cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT). This deletion happens in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which involves the last cytosine (C) of isoleucine 507 (isoleucine 507ATC) and the two thymidine oligonucleotide (T) of phenylalanine 508 (phenylalanine 508TTT) codons. The significances of this important deletion are the deletion of phenylalanine at the 508 position of the cystic fibrosis transmembrane conductance regulator protein (ΔF508), an identical codon modification for isoleucine 507 (isoleucine 507ATT), and protein dysfunction. Materials and methods Fluorescence and ultraviolet−visible thermal studies were performed for WT and mutant-type target full systems. The target DNAs used were in the form of short oligonucleotides. The tandem probes system was used for detection of WT and single-nucleotide polymorphism alleles of human 3-bp ΔF508 (TTT) homozygous deletion. The pyrene dye attached to a probe oligonucleotide (15 mer) undergoes an excimer fluorescence intensity change on hybridization of the two probes to the WT compared with mutant-type targets. Results Our results indicate that the system consisting of the target sequence and the two probe oligonucleotides bearing the pyrene dye assemble correctly at the specified target. Once the full system (two probes and target) is arranged under suitable conditions, a red-shift emission and change in fluorescence intensity are seen at an excimer wavelength of 480 nm. Thermal studies also showed significant differences in Tm between mutated and unmutated CF genes. The results suggest that the differences in the fluorescence intensity at 480 nm and the spectrophotometric Tm (s) for the mutated and unmutated CF gene can be attributed to the type of binding of the probe to the target. Conclusion On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best deletion discrimination detection ability. The system was sensitive to deletion nucleotide polymorphisms and this may help in high-throughput applications in genetic testing and molecular diagnostics.