Progress towards single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA

The enzymatic incorporation of modified dNTPs into a growing DNA strand has intensively been studied. Modifications were detectable reporter groups su ch as digoxigenin or biotin, fluorochromes or aliphatic side chains covalen tly attached to the base. Incorporation efficiencies were determined with s everal DNA polymerases using linear primer-extension reactions followed by denaturing PAGE as a high-resolution detection system. We describe the enzy matic synthesis of DNA consisting of modified nucleotides exclusively. A de fined template-primer system allows us to trace incorporation: (1) in up to 18 neighboring positions for several dUTP-derivatives; or (2) in stretches of DNA of up to 40 bases in length with complete substitution of all four natural dNTPs by differently modified counterparts. Synthesized DNA molecul es are shown to particularly exhibit dramatically altered physico-chemical properties by contrast with native DNA. These results provide a fundamental data set for probe generation in single-molecule DNA sequencing (SMS). (C) 2001 Elsevier Science B.V. All rights reserved.