Researchers in the Columbia Genome Center and the Department of Chemical Engineering of Columbia University developed a novel approach for constructing a large number of combinatorial fluorescence energy transfer (CFET) tags by exploiting energy transfer and combinatorial synthesis to tune the fluorescence emission signatures for multiplex genomics analysis. All of the CFET tags can be excited at a single wavelength and analyzed by a simple optical system. In the August 1 issue of Nature Biotechnology, Dr. Jingyue Ju, associate professor of chemical engineering and head of DNA sequencing and chemical biology at Columbia Genome Center, and researchers Anthony K. Tong, Zengmin Li, Gregg S. Jones and James J. Russo report this new development.

Fluorescent labels are widely used in biomedical research, including DNA sequencing, genetic variation analysis and gene expression measurements. The first generation of energy transfer fluorophores co-invented by Ju in 1993 played a major role in sequencing the human genome. Yet the development of fluorescence detection for multiplexed applications has been hindered by the limited number of available fluorophores. Ju and his colleagues report that a small number of individual fluorophores can be used to construct a larger number of fluorescent oligonucleotide tags. Eight CFET tags with unique fluorescence signatures, detected by the multi-color fluorescence DNA analyzer MegaBACE 1000, were constructed using one to three individual fluorescent dyes. A 1', 2'-dideoxyribose phosphate spacer was used to separate the donor/acceptor to tune the ET efficiency, thereby generating the unique fluorescence signatures. The spacer was also used as an electrophoretic mobility tag to tune the mobility of the CFET-labeled DNA for multiplex single nucleotide polymorphisms (SNPs) detection. Multiple SNPs were identified simultaneously using a library of CFET tags on synthetic DNA templates as well as a PCR product from the tumor suppressor retinoblastoma gene. This research established the feasibility that a larger library of CFET tags can be constructed using more than three different chromophores by designing different separation distances between the chromophores, and using appropriate detection channels in the optical system for measuring the unique fluorescence signatures for multiplex genomics analysis.

The research was funded by the National Science Foundation (Biophotonics Partnership Initiative Grant 86933) and the Strategic Initiative Program of the Office of the Provost at Columbia, the Columbia Genome Center as well as instrumentation support from Amersham Pharmacia Biotech, Inc.