Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization

Dongjian Cao,Caili Xi,Qingming Luo,Zhihong Zhang,Kaiheng Zhu,Sa Wu,Jie Yang,Hui Gong,Dong Li

doi:10.1038/s42003-021-02762-2

Abstract

Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs are used to develop AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows.

Highlights

Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues
The spatial and quantitative information of nucleic acids can be detected in the cells or tissues under a fluorescent microscope using single-molecule RNA fluorescence in situ hybridization imaging, which allows visualizing the spatial distribution of transcripts through hybridization of target genes[6]
Our results showed that AmpFISH has several advantages: (1) up to 100-fold signal-to-noise amplification; (2) simultaneous detection of FISH signals with fluorescent proteins or immunofluorescence (IF) in tissues; (3) simple workflow; and (4) cost-efficiency

Summary

Introduction

Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Fluorescence in situ hybridization (FISH), a powerful tool, allows the detection of nucleic acid molecules within the intact structure and in the morphology of cells or tissue sections. The spatial and quantitative information of nucleic acids can be detected in the cells or tissues under a fluorescent microscope using single-molecule RNA fluorescence in situ hybridization (smFISH) imaging, which allows visualizing the spatial distribution of transcripts through hybridization of target genes[6]. Our results showed that AmpFISH has several advantages: (1) up to 100-fold signal-to-noise amplification; (2) simultaneous detection of FISH signals with fluorescent proteins or immunofluorescence (IF) in tissues; (3) simple workflow; and (4) cost-efficiency. We present a convenient and versatile tool for FISH detection, providing a promising application for transcript and chromosome assays in the cells or in complex and heterogeneous tissues

Methods

Results

Conclusion