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.
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.
Bridgman
A J
AJ
Department of Biochemistry and Centre for Gene Research, University of Otago, Dunedin, New Zealand.
Petersen
G B
GB
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry*; D017931:DNA Primers; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis*; D004587:Electrophoresis, Agar Gel; D007202:Indicators and Reagents; D008628:Mercury; D008767:Methylmercury Compounds / Q000138:chemical synthesis*; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D012680:Sensitivity and Specificity
D017931:DNA Primers; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D008767:Methylmercury Compounds; C102406:mercurated dUTP; C027078:deoxyuridine triphosphate; D004247:DNA; D008628:Mercury