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Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry.


ABSTRACT: We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ?70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue).

SUBMITTER: Vranken C 

PROVIDER: S-EPMC3985630 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry.

Vranken Charlotte C   Deen Jochem J   Dirix Lieve L   Stakenborg Tim T   Dehaen Wim W   Leen Volker V   Hofkens Johan J   Neely Robert K RK  

Nucleic acids research 20140121 7


We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequenci  ...[more]

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