|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
20. Development of a Multilabel DNA Mapping Technique Using SERS Gene Probes
Tuan Vo-Dinh1, David
L. Stokes1, Guy D. Griffin1, Jean-Pierre Alarie1,
Edward J. Michaud1, Terry Bunde1, Ung-Jin Kim2,
Melvin I. Simon2
We report the development of a novel approach for use in DNA mapping and bacterial artificial chromosomes (BAC) colony hybridization using a unique type of DNA gene probe based on surface-enhanced Raman scattering (SERS) labels. An important step toward sequencing the human genome involves assembling ordered, overlapping sets (contigs) of clones that have been mapped and well characterized. A unique approach that would greatly facilitate large-scale genomic sequencing involves building genome-wide BAC-based contig maps.
In this work, we have developed various types of SERS-active substrates that can be used to provide this "label-multiplex" capability, thereby reducing the time for genome characterization. We have developed various schemes for binding SERS labels onto DNA targets for use in BAC clone mapping. We have demonstrated the feasibility of a multi-label SERS detection scheme, whereby multiple labels can be detected simultaneously in each multiplex probing cycle. Raman spectroscopy is an important analytical tool due to its excellent specificity for chemical group identification. With the use of the SERS effect, Raman scattering efficiency can be enhanced by factors of up to 108 when a compound is adsorbed on or near special metal substrates1. The surface-enhanced Raman gene (SERGen) probes do not require the use of radioactive labels and have a great potential to provide both sensitivity and selectivity for DNA mapping and sequencing. The method is aimed at simultaneous detection of multiple probes for DNA mapping/sequencing using BAC clone applications. The technology is designed to be versatile and broad-based, and to allow a rapid and shortcut approach to significantly improve the speed of BAC colony hybridization. cDNAs are an excellent resource to rapidly build genome wide BAC contigs. They represent inexpensive, efficient probes to screen BAC libraries by colony hybridization. However, the conventional approach relying on 32P-labeled probes is laborious and time consuming. Multiplexing probes with non-radiative chemicals that can be efficiently distinguished after hybridization will greatly reduce the time and effort for establishing the large-scale BAC library that is required for characterizing the entire genome of human, mouse and other organisms.
This research is sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy under contract DE AC05-960R22464 with Lockheed Martin Energy Research Corporation.
1T. Vo-Dinh, "Surface-enhanced Raman spectroscopy using metallic nanostructures" in Trends in Analytical Chemistry, 17, 557 (1998)
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