Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization.
Dongjian CaoSa WuCaili XiDong LiKaiheng ZhuZhihong ZhangHui GongQingming LuoJie YangPublished in: Communications biology (2021)
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.
Keyphrases
- single molecule
- living cells
- label free
- circulating tumor
- atomic force microscopy
- nucleic acid
- gene expression
- induced apoptosis
- climate change
- loop mediated isothermal amplification
- real time pcr
- signaling pathway
- oxidative stress
- quantum dots
- cell death
- photodynamic therapy
- cell cycle arrest
- energy transfer
- high speed
- pi k akt
- molecularly imprinted