Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications

Abstract

Fluorescence in situ hybridization (FISH) is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes, and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains, and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbias and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH) allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH) using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells DNA and RNA FISH techniques have visualized intra-nuclear genomic structure and sub-cellular transcriptional dynamics of many genes and revealed their functions in various biological processes.

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…Working principles of common hybridization‐based probes. A) A schematic of linear antisense probes, whereby a dye‐labeled recognition sequence binds to its complementary target illustrated in the same color . B) Linear FRET probes involve hybridization of two different linear antisense probes to adjacent regions of a target sequence, bringing a FRET pair into proximity that results in a fluorescence signal that can be monitored .…”

“…Many techniques for cellular analysis such as northern blotting, western blotting, fluorescence in situ hybridization (FISH), RT‐PCR, DNA microarrays, and electron microscopy rely on the fixation or lysis of cells . Moreover, as the amount of material from one cell is often insufficient for accurate analysis, a bulk population of cells is used for methods that analyze cell lysates.…”

“…The last broad classification of detection scenarios concerns the ability to quantify targets intracellularly, in contrast to simply detecting or knowing relative amounts. FISH has widely been used for absolute quantification of transcript numbers in fixed cells . Methods for quantitative probing in live cells usually incorporate a reference probe/dye as an internal control to which the signal from the targeting probe is normalized.…”

“…[2][3][4][5] Many techniques for cellular analysis such as northern blotting, western blotting, fluorescence in situ hybridization (FISH), RT-PCR, DNA microarrays, and electron micro scopy rely on the fixation or lysis of cells. [5][6][7][8][9][10][11] Moreover, as the amount of material from one cell is often insufficient for accurate analysis, a bulk population of cells is used for methods that analyze cell lysates. Information about dynamics of various molecules inside cells and cell-to-cell heterogeneity is often lost in such cases due to ensemble averaging.…”

Chromosome Imbalances in Cancer: Molecular Cytogenetics Meets Genomics

“…Technical innovation in cytogenetic analyses is still increasing thanks to the development of novel fluorescent probes with improved sensitivity, high-resolution microscopy, and methods to study single cells or single molecules [Speicher and Carter, 2005;Cui et al, 2016]. This is causing revision and refinement of the principles necessary to understand the mechanisms of the origin of chromosome rearrangements in cancer, their eventual causative role, their possible use as biomarkers of cancer evolution or to predict response to therapy [Mertens et al, 2015;Weckselblatt and Rudd, 2015;Willis et al, 2015].…”