Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry.
Nucleic Acids Res
; 42(7): e50, 2014 Apr.
Article
en En
| MEDLINE
| ID: mdl-24452797
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 â¼70% 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).
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
Metilasas de Modificación del ADN
/
Genómica
/
Química Clic
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2014
Tipo del documento:
Article
País de afiliación:
Dinamarca
Pais de publicación:
Reino Unido