Molecular Beacons: Signalling Nucleic Acid Probes, Methods, and Protocols

Chapter 1 Larry E. Morrison Basic Principles of Fluorescence and Energy Transfer Fluorescence is highly sensitive to environment, and the distance separating fluorophores and quencher molecules can provide the basis for effective homogeneous nucleic acid hybridization assays. Molecular interactions leading to modulation of fluorescence include collisions, ground state and excited state complex formation, and long-range dipole-coupled energy transfer. These processes are well understood and equations are provided for estimating the effects of each process on fluorescence intensity. Estimates for the fluorescein-tetramethylrhodamine donor-acceptor pair reveal the relative contributions of dipole-coupled energy transfer, collisional quenching, and static quenching in several common assay formats, and show that the degree of quenching is dependent upon the hybridization complex formed and the manner of label attachment. Applications Chapter 2 David S. Perlin,* Sergey Balashov, Steven Park Multiplex Detection of Mutations Rapid and reliable detection of mutations at the genetic level is an integral part of modern molecular diagnostics. These mutations can range from dominant single nucleotide polymorphisms within specific loci to co-dominant heterozygotic insertions and they present considerable challenges to investigators in developing rapid nucleic acid based amplification assays that can distinguish wild-type from mutant alleles. The recent improvements of real time PCR using self reporting fluorescence probes have given researchers a powerful tool in developing assays for mutation detection that can be multiplexed for high throughput screening of multiple mutations and cost effectiveness. Here we describe an application of a multiplexed real time PCR assay using Molecular Beacon probes for the detection of mutations in codon 54 of the CYP51A gene in Aspergillus fumigatus conferring triazole resistance. Chapter 3 Arvind A. Bhagwat*, Jitu Patel, Trina Chua, Audrey