DESIGN AND SYNTHESIS OF FLUORESCENCE ENERGY-TRANSFER DYE-LABELED PRIMERS AND THEIR APPLICATION FOR DNA-SEQUENCING AND ANALYSIS

We have designed and synthesized fluorescent oligonucleotide primers h aving improved fluorescence and electrophoretic properties by exploiti ng the concept of resonance fluorescence energy transfer (ET), These p rimers carry a fluorescein derivative at the 5' end as a common fluore scence donor and other fluorescein and rhodamine derivatives attached to a modified thymidine within the primer sequence as accepters. These primers all have strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm . The fluorescence emission intensity of the ET primers increases as t he spacing between the donor and accepters is increased, and of the sp acings studied the strongest fluorescence was observed when the number of nucleotides between the donor and accepters is 10. The electrophor etic mobilities of the primers were also found to be a function of the spacing between the donor and the accepters, and the mobilities of th e single base extension DNA fragments generated with primers F10F, F10 J, F10T, and F10R were closely matched, With excitation at 488 nm, the fluorescence of the four optimized ET primers (F10F, F10J, F10T, and F10R) is 2- to 14-fold greater than that of the corresponding primers labeled with only one dye. The increased fluorescence intensity of the ET primers and the substantially similar mobilities of the DNA fragme nts generated with the four ET primers allow four color DNA sequencing on a capillary electrophoresis DNA sequencer using a single laser lin e at 488 nm for excitation and without applying mobility shift adjustm ents, With single-stranded M13mp18 DNA as the template, a typical run with the ET primers on a commercial sequencer provided DNA sequences w ith 99-100% accuracy in the first 500 bases using 8-fold less DNA temp late than that typically required using T7 DNA polymerase. (C) 1995 Ac ademic Press, Inc.