Jingyue Ju,* Indu Kheterpal, Su-Chun Hung[1], James R. Scherer, Alexander N. Glazer[1] and Richard A. Mathies
Department of Chemistry, University of California, Berkeley, CA 94720.
Our goal is to develop novel fluorescently labeled primers for DNA sequencing and multiplex genetic analysis that have enhanced fluorescence intensities and improved match in electrophoretic mobilities.[1,2] From a library of energy transfer (ET) fluorescent primers (M13, -40) with varying donor and acceptor separation distances which we developed previously, we chose four ET primers (F1OF, F1OJ, F1OT and F1OR) which satisfied the above criteria as a representative optimized set for 4-color DNA sequencing [3]. The fluorescence of these four ET primers is 2- to 14-fold greater than that of the corresponding primers labeled with only one dye when excited at 488 nm. The increased fluorescence intensity of the ET primers and the substantially similar mobilities of the DNA fragments generated with the four ET primers allow four-color DNA sequencing on a capillary array electrophoresis (CAE) DNA sequencer using a single laser line at 488 nm for excitation, and without applying mobility shift adjustments. With single-stranded M13mpl8 DNA as the template, a typical run with the ET primers on a commercial ABI 373 sequencer provided DNA sequences with 99%-100% accuracy in the first 500 bases using 8-fold less DNA template (0.25 µg) than that typically required using T7 DNA polymerase. In further work, we have developed an ET cassette which can be used to label primers of any sequence, thereby significantly extending the utility of energy transfer labels [4]. We have also found that 5 or 6-carboxyrhodamine-6G can be used in ET primers to replace JOE with improved match in the mobility shift and no change in fluorescence sensitivity [5]. The design and synthesis of the ET primers and their application to high-throughput CAE DNA sequencing will be presented.
Supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-FG-91-61125. Support from Amersham Life Science Inc. is also acknowledged.
*DOE Human Genome Distinguished Postdoctoral fellow
[1] Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
[1]. J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies. Proc. Natl. Acad. Sci. 92, 4347-4351 (1995)
[2]. Y. Wang, J. Ju, B. A. Carpenter, J. M. Atherton, R. A. Mathies and G. F. Sensabaugh. Anal. Chem. 67, 1197- 1203 (1995)
[3]. J. Ju, I. Kheterpal, J. R. Scherer, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies. Anal. Biochem. 231, 131-140 (1995)
[4]. J. Ju, A. N. Glazer and R. A. Mathies, Universal Energy Transfer Cassette-labeled Primers for DNA Sequencing and Analysis, in preparation.
[5]. S-C. Hung, J. Ju, R. A. Mathies and A. N. Glazer, Energy Transfer Primers with 5 or 6. Carboxyrhodamine-6G as Acceptor Chromophores, in preparation.