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DNA fluorocode: A single molecule, optical map of DNA with nanometre resolution

2010; Royal Society of Chemistry; Volume: 1; Issue: 4 Linguagem: Inglês

10.1039/c0sc00277a

2041-6539

Robert K. Neely, Peter Dedecker, Jun‐ichi Hotta, Giedrė Urbanavičiūtė, Saulius Klimašauskas, Johan Hofkens,

Genomics and Chromatin Dynamics

We present a new method for single-molecule optical DNA mapping using an exceptionally dense, yet sequence-specific coverage of DNA with a fluorescent probe. The method employs a DNA methyltransferase enzyme to direct the DNA labelling, followed by molecular combing of the DNA onto a polymer-coated surface and subsequent sub-diffraction limit localization of the fluorophores. The result is a 'DNA fluorocode'; a simple description of the DNA sequence, with a maximum achievable resolution of less than 20 bases, which can be read and analyzed like a barcode. We demonstrate the generation of a fluorocode for genomic DNA from the lambda bacteriophage using a DNA methyltransferase, M.HhaI, to direct fluorescent labels to four-base sequences reading 5′-GCGC-3′. A consensus fluorocode that allows the study of the DNA sequence at the level of an individual labelling site can be generated from a handful of molecules.