DNA Sequence 1996-01-01

An improved method for the synthesis of mercurated dUTP. Enzymic synthesis of Hg-labelled DNA of high molecular weight suitable for use in an image based DNA sequencing strategy.

A J Bridgman, G B Petersen

Index: DNA Seq. 6 , 199-209, (1996)

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Abstract

The development of high-resolution scanning-probe microscopes has reawakened interest in the possibility of sequencing large nucleic acid molecules by direct imaging. Such an approach would be facilitated by the availability of effective methods for increasing contrast by labelling specific nucleotides, and the utility of introducing mercury atoms into complete DNA molecules through the enzymic polymerisation of mercurated pyrimidine deoxynucleoside triphosphates has been re-investigated. A simplified and improved method for the synthesis of a heat- and thiol-stable, mercurated derivative of deoxyuridine triphosphate in high yield and the incorporation of this precursor into full-length copies of a single-stranded phage M13 template are described. The DNA product has been fully characterised and the quantitative and specific replacement of thymidylic acid residues by the mercurated analogue demonstrated.