Oligonucleotide probes for RNA-targeted fluorescence in situ hybridization.

The need for accurate and rapid methodology for detecting cells in environmental and clinical samples has led to the development of in situ detection methods, where fixed or intact cells can be imaged directly. In this chapter, we focus on the use of labeled oligonucleotide probes in fluorescence in situ hybridization (FISH). We give an overview of FISH probe design, covering issues of affinity and specificity of probes, probe backbone options, cellular targets, and accessibility of target sequences. Decisions that must be made to design optimal probes are evaluated, and available resources to assist in probe design, such as secondary structure, Tm calculation, and site accessibility software, are discussed. We cover different types of FISH probes that have been reported in the recent literature, including standard fluorescently labeled oligonucleotide probes and newer classes of quenched oligonucleotide probes: molecular beacons and quenched autoligation probes. Advantages and disadvantages of the different probe types are examined and recent literature applications are discussed. The current state of the art in the field as well as limitations and challenges in detection are evaluated.