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Issue 3, 2019
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Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

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Abstract

The recent advances in the single cell genome analysis are generating a considerable amount of novel insights into complex biological systems. However, there are still technical challenges because each cell has a single copy of DNA to be amplified in most single cell genome analytical methods. In this paper, we present a novel approach to directly visualize a genomic map on a large DNA molecule instantly stained with red and green DNA-binding fluorescent proteins without DNA amplification. For this visualization, we constructed a few types of fluorescent protein-fused DNA-binding proteins: H-NS (histone-like nucleoid-structuring protein), DNA-binding domain of BRCA1 (breast cancer 1), high mobility group-1 (HMG), and lysine tryptophan (KW) repeat motif. Because H-NS and HMG preferentially bind A/T-rich regions, we combined A/T specific binder (H-NS-mCherry and HMG-mCherry as red color) and a non-specific complementary DNA binder (BRCA1-eGFP and 2(KW)2-eGFP repeat as green color) to produce a sequence-specific two-color DNA physical map for efficient optical identification of single DNA molecules.

Graphical abstract: Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

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Supplementary files

Article information


Submitted
27 Jul 2018
Accepted
07 Oct 2018
First published
08 Oct 2018

Analyst, 2019,144, 921-927
Article type
Paper

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

J. Park, S. Lee, N. Won, E. Shin, S. Kim, M. Chun, J. Gu, G. Jung, K. Lim and K. Jo, Analyst, 2019, 144, 921
DOI: 10.1039/C8AN01426D

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