Combinatorial Fluorescence Energy Transfer Tags with Phosphate Spacers: New Molecular Tools for the Genomics Revolution

摘要

The massive information generated by the Human Genome Project drives the need for multiplex fluorescent tags that can be used to study many biological targets simultaneously. However, due to the limitations of the spectral region and therefore the availability of the optical detectors, the number of available fluorescent dyes that have distinguishable emission spectra is limited. Previously, we exploited the principle of fluorescent energy transfer (ET) to enhance fluorescence emission for the successful development of four ET tags for DNA sequencing that are widely used in the Human Genome Prroject. Recently, we developed a novel approach for assembling a large number of combinatorial fluorescence energy transfer (CFET) tags by exploiting energy transfer (ET) and combinatorial synthesis to tune the fluorescence emission signatures for multiplex biological assays. All of the CFET tags can be excited at a single wavelength and analyzed by a simple optical system. As an example, eight CFET tags with unique fluorescence signatures, detected by a three-color capillary array electrophoresis system with 488 nm excitation, were constructed using one to three individual fluorophores. 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. We present here the design, synthesis and application of the CFET tags for identifying multiple genetic mutations using ligase reaction.