FLUORESCENCE ENERGY-TRANSFER DYE-LABELED PRIMERS FOR DNA-SEQUENCING AND ANALYSIS

Fluorescent dye-labeled DNA primers have been developed that exploit f luorescence energy transfer (ET) to optimize the absorption and emissi on properties of the label, These primers carry a fluorescein derivati ve at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the prime r sequence as accepters. Adjustment of the donor-acceptor spacing thro ugh the placement of the modified thymidine in the primer sequence all owed generation of four primers, all having strong absorption at a com mon excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm, The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities b y gel electrophoresis, With argon-ion laser excitation, the fluorescen ce of the ET primers and of the DNA sequencing fragments generated wit h ET primers is 2- to 6-fold greater than that of the corresponding pr imers or fragments labeled with single dyes, The higher fluorescence i ntensity of the ET primers allows DNA sequencing with one-fourth of th e DNA template typically required when using T7 DNA polymerase, With s ingle-stranded M13mp18 DNA as the template, a typical sequencing react ion with ET primers on a commercial sequencer provided DNA sequences w ith 99.8% accuracy in the first 500 bases. ET primers should be genera lly useful in the development of other multiplex DNA sequencing and an alysis methods.