Introduction to the Workshop
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Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Luminescent Lanthanide Ions as Labels for DNA Sequencing and Mapping

Gilbert M. Brown[1], Jeffrey E. Elbert[1], Frederick V. Sloop[2], Mitchel J. Doktycz[3], Richard A. Sachleben[1], and K. Bruce Jacobson[3]
Chemical and Analytical Sciences Division,[1] Biology Division,[2] and Health Sciences Research Division,[3] Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831-6119

The goal of this research program is the development of a new luminescent labeling system for DNA that will be a successor to the fluorescent organic dyes currently used for sequencing. This labeling system can also be used with hybridization probes for DNAs attached to nylon or nitrocellulose membranes (Southern Blot). Luminescence of the lanthanide ions Sm(III), Eu(III), Tb(III), and Dy(III) has narrower bandwidths than the fluorescent organic compounds currently used to label DNA, and these narrow linewidths will allow multiple probes to be detected simultaneously with little overlap of emission and potentially to get greater resolution in the bands. Furthermore, the long lifetimes allow detection with a lower background. A derivative of the macrocyclic chelating agent, 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid (DOTA), is used to attach the lanthanide [Ln(III)] ions to oligonucleotides, and this ligand forms stable, kinetically inert complexes with these metal ions. The ligand, as an isothiocyanate derivative, is reacted with a hexylamine linker arm on an oligonucleotide to generate a labeled primer for the Sanger sequencing procedure or PCR. Detection sensitivity for luminescence from Ln(III) ions can be greatly enhanced if excitation is to a ligand based state having a long lived triplet electronic state. A naphthylmethyl derivative of DOTA and an acetophenone derivative of DO3A (tetraazacyclododecanetriacetic acid) were prepared and the sensitizer groups served as antennas to funnel excitation energy to complexed Ln(III) ions. The benzene ring of the acetophenone derivative is being nitrated so that upon reduction the resulting amine will provide an attachment point to DNA. A third derivative, the benzylisothiocyano derivative of tetraazacyclododecane with three acetate arms and an acetophenone antenna has been prepared and characterization is in progress. The chemistry for attachment of the ligand to oligonucleotides is being developed so that it can be added as the last step in the automated synthesis of the oligonucleotide while still attached to a solid support. Detection limits for oligonucleotides, labeled with the Ln(III) reagents, were determined following capillary electrophoresis with on-column detection using a pulsed laser and time-gated luminescence detection. Work is in progress toward preparing and testing an M13 primer, labeled with an Eu(III) complex having a sensitizing antenna group, for use in the Sanger sequencing procedure. Initial sequencing experiments will be carried out using the well studied M13mp18 system. We will utilize two-color labeling using Eu(III) and Tb(III) complexes with capillary electrophoresis to separate the DNA fragments and two filter-photomultiplier tube assemblies for luminescence detection. These results will guide development of a second generation instrument which will have a polychromator/CCD detector for simultaneous detection of all four Ln(III) ions.

This research was sponsored by the Office of Health and Environmental Research, U. S. Department of Energy, under contract No. DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc. This research was sponsored in part by the appointment of J. E. Elbert to a Human Genome Distinguished Postdoctoral Fellowship sponsored by the U. S. Department of Energy, Office of Health and Environmental Research, and administered by the Oak Ridge Institute for Science and Energy.