Antibody–nucleotide conjugate as a substrate for DNA polymerases

Abstract

Here we report on the development of an antibody-modified nucleotide and its sequence-selective incorporation into nascent DNA catalysed by DNA polymerases. Although the modification of the nucleotide is several orders of magnitude larger than the natural dNTP substrate and even exceeds the size of the DNA polymerase, it is well accepted by the enzyme. Moreover, the recognition of the antibody is not abolished by the conjugation but can be recognized by a secondary antibody that is conjugated to a signal-generating enzyme (i.e., horse radish peroxidase). This product can thus be exploited for a colorimetric read-out of nucleotide incorporation by the naked eye that allows detection of DNA as low as 10 amol. In future, assays like the one described herein might allow nucleic acid diagnostics at single nucleotide resolution without any laboratory equipment.