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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Published on: November 22, 2014

Determination of specific DNA sequences and their hybridisation processes by elemental labelling followed by

Lucía López-Fernández¹, Elisa Blanco-González, Jörg Bettmer

¹University of Oviedo, Department of Physical and Analytical Chemistry, C/Julián Clavería 8, 33006 Oviedo, Spain. bettmerjorg@uniovi.es.

|May 20, 2014

Abstract:

Detection of specific DNA sequences is nowadays an important tool in many scientific areas such as forensic science or clinical diagnosis. Although numerous approaches have been suggested for this challenging analysis, certain limitations still remain. In order to overcome these disadvantages, novel and alternative methodologies are required. In this work, we present a strategy based on elemental (lanthanide) labelling of DNA probes followed by size-exclusion chromatography (SEC) coupled with inductively coupled plasma-mass spectrometry (ICP-MS) for monitoring and determining complementary oligonucleotide sequences (oligonucleotide targets) and for visualising the corresponding hybridisation processes. The synthesis and characterisation of the DNA probes are described in detail. SEC was found to be suitable to discriminate between the DNA probe and the hybridised product. Using labelling of different probes with different lanthanides, multiplexed detection of the sought DNA sequences was possible as demonstrated here on three DNA probes (derivatised with Eu, Tb, and Ho, respectively). The achievable detection limits were in the range between 5 and 11 fmol absolute.

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