Colorimetric allele analysis based on the DNA-directed cooperative formation of luminous lanthanide complexes
Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan.Nucleic Acids Symposium Series 02/2006; 50(50):105-6. DOI: 10.1093/nass/nrl052
We present the facile technique of colorimetric SNP analysis through DNA-templated cooperative complexation between a luminescent lanthanide ion (Ln(3+): Tb(3+) or Eu(3+)) and two ODN (oligodeoxyribonucleotide) conjugates carrying a metal chelator. Ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthrorine (phen) were covalently attached to ODNs to form the conjugate probes, capture and sensitizer probes, respectively. The sequences of the conjugates were designed so as to form a tandem duplex with a target with their auxiliary units facing each other, providing a microenvironment to accommodate Ln(3+). The capture probes for the wild-type (wt) and the mutant (mut) of thiopurine S-methyltransferase gene, were mixed with equimolar amounts of Tb(3+) and Eu(3+), respectively. Then both of the allele specific capture probe solutions and the sensitizer probe were added to three different solutions containing the targets, wt/wt (G/G), mut/mut (C/C), and wt/mut (G/C). The solutions emitted in green, red, and yellow, respectively; the colors were identified even by the naked eye.
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ABSTRACT: We present the facile technique of colorimetric SNP (single nucleotide polymorphism) analysis through DNA-templated cooperative complexation between a luminescent lanthanide ion (Ln(III): Tb(III) or Eu(III)) and two ODN (oligodeoxyribonucleotide) conjugates carrying a metal chelator. Families of complexane-type chelators and heterocyclic aromatic ligands were covalently attached to ODNs to form conjugates for application as capture and sensitizer probes. The sequences of the conjugates were designed so as to form a ternary tandem duplex with the target, where their auxiliary units face each other, providing a microenvironment to accommodate Ln(III). Only the combination of EDTA (ethylenediaminetetraacetic acid) conjugates and phen (1,10-phenanthroline) conjugates provided significant emissions with quantum yields of 3.3% and 1.5% for Tb(III) and Eu(III), respectively, in the presence of the target. Biallelic polymorphism in the TPMT (thiopurine S-methyltransferase) gene, wt/wt (G/G), mut/mut (C/C), and wt/mut (G/C), were distinguished as emissions in green, red, and yellow, respectively; the colors were identified even by the naked eye.
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ABSTRACT: The sensitivity and high resolution of optical bioprobes allied to the specific advantages of lanthanide ions have stirred hefty interest during the past two decades in view of the need for more sensitive in vitro analyses and for better and faster in vivo imaging. As a result, lanthanide luminescent bioprobes (LLBs) have been the subject of sustained attention, particularly when coupled with fast developing techniques such as time-resolved microscopy, multi-photon excitation, FRET, lab-on-a-chip analysis, and nanobiotechnology. In this review, the basic properties of lanthanide luminescence are first summarized, along with the main features prevailing to the design of bioprobes and bioconjugates. A broad overview of the numerous fields of applications of LLBs is then given, spanning applications from immunoassays to analytical biosensors for in cellulo determination of pH, p(O 2 ), hydrogen peroxide, enzyme activity, DNA content and hybridization, among others. Cell, tissue, and small animal imaging are also described, as well as multiplex detection of biomarkers expressed by cancerous cells and tissues. In all these applications, lanthanide optical probes present definite advantages over conventional methods of analysis, not only from the sensitivity point of view but also from an economical standpoint. Future developments in bio-analysis and medical diagnosis will make a large use of these probes.