An Application-Directed, Versatile DNA FISH Platform for Research and Diagnostics.

Abstract

DNA fluorescence in situ hybridization (DNA FISH) has emerged as a powerful microscopy technique that allows a unique view into the composition and arrangement of the genetic material in its natural context-be it the cell nucleus in interphase, or chromosomes in metaphase spreads. The core principle of DNA FISH is the ability of fluorescently labeled DNA probes (either double- or single-stranded DNA fragments) to bind to their complementary sequences in situ in cells or tissues, revealing the location of their target as fluorescence signals detectable with a fluorescence microscope. Numerous variants and improvements of the original DNA FISH method as well as a vast repertoire of applications have been described since its inception more than 4 decades ago. In recent years, the development of many new fluorescent dyes together with drastic advancements in methods for probe generation (Boyle et al., Chromosome Res 19:901-909, 2011; Beliveau et al., Proc Natl Acad Sci U S A 109:21301-21306, 2012; Bienko et al., Nat Methods 10:122-124, 2012), as well as improvements in the resolution of microscopy technologies, have boosted the number of DNA FISH applications, particularly in the field of genome architecture (Markaki et al., Bioessays 34:412-426, 2012; Beliveau et al., Nat Commun 6:7147, 2015). However, despite these remarkable steps forward, choosing which type of DNA FISH sample preparation protocol, probe design, hybridization procedure, and detection method is best suited for a given application remains still challenging for many research labs, preventing a more widespread use of this powerful technology. Here, we present a comprehensive platform to help researchers choose which DNA FISH protocol is most suitable for their particular application. In addition, we describe computational pipelines that can be implemented for efficient DNA FISH probe design and for signal quantification. Our goal is to make DNA FISH a versatile and streamlined technique that can be easily implemented by both research and diagnostic labs.