From probe design to applications in clinical settings, this book provides a diverse set of instructive examples, guided by experts in the field who offer easy-to-follow experimentals. The book first offers an introduction to the basic principles of fluorescence and then describes applications of fluorogenic probes in real-time PCR, which currently is the gold standard for quantitative DNA and RNA analysis.
Coverage extends the potential of realtime as well as advocates simplifications of the probe technologies. It also presents a new simplified molecular beacon design, EasyBeacons, and demonstrates the utility in DNA methylation profiling.
Table of ContentsChapter 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 Chan, Saúl Ruiz Cruz, and Gustavo A. González Aguilar Detection of Salmonella species in foodstuffs† Conventional methods to detect Salmonella spp. in foodstuffs may take up to one week. Considering the limited shelf life of ready-to-eat foods as well as fresh produce, rapid methods for pathogen detection are required. Real-time detection of Salmonella spp. will broaden our ability to screen large number of samples in a short time. This chapter describes a step-by-step procedure using an oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (Molecular Beacon; MB) in a real-time polymerase chain reaction (PCR) assay. The capability of the assay to detect Salmonella species from artificially inoculated fresh- and fresh-cut produce as well as resdy-to-eat meats is demonstrated. The method uses internal positive and negative controls which enable researchers to detect false-negative PCR results. The procedure uses the buffered peptone water for the enrichment of Salmonella spp. and successfully detects the pathogen at low level of contamination (2-4 cells/25 g) in less than 24 h. Chapter 4 Gunter Schmidtke and Marcus Groettrup Identification of homozygous transgenic mice by genomic real time PCR The 26S proteasome is the executing protease of the ubiquitin dependent degradation system. It consists of one or two 19S regulatory sub complexes and one 20S proteolytic sub complex (1). The 20S proteasome is a barrel shaped cylinder which consists or four stacked rings (2). Each of the two outer rings consists of 7 different a-subunits, whereas each of the two inner rings is formed by 7 different b-subunits (3). Only three of these b-subunits bear a catalytically active N-terminal threonine (4,5). Under normal conditions these are b1(delta), b2 (Z) and b5 (mb1).