New Dyes for DNA Sequencing 



Hee-Chol Kang. James E. Whiiaker. Peter C. Hewitt, and Richard P. Haugland 



Molecular Probes, Inc.. Eugene, OR 97402 



(415)486-5717 



We have been actively synthesizing and evaluating sets of new tluorophores that can be 

 excited by the argon laser at 488 or 5 14 nm for possible use in DNA sequencing. The 

 objectives are to synthesize sets of four dyes whose emission spectra have relatively low 

 overlap, whose fluorescence when excited with the argon laser is brighter than currently 

 available tluorophores. and whose properties of ionic charge are uniform for minimum 

 interference with electrophoretic separations. Principal among the dyes prepared have 

 been tluoreNcein-rhodamine bifluorophores in which the energy absorbed by the 

 fluorescein is emitted almost totally at the rhodamine emission wavelength. Examples 

 of these dyes have been prepared where the energy transfer has been >98% efflcient 

 with pseudo-Stokes shifts of up to 100 nm. Several reactive versions of rhodamine and 

 of rhodol dyes have been prepared which fluoresce when excited by the argon laser and 

 whose emission is brighter than tetramethylrhodamine. The fourth class of new 

 fluorophores with potential for use in DNA sequencing are reactive, boron dipyrro- 

 methene difluoride (Bodipy"^' ) derivatives, which have been prepared in several 

 reactive fonns. Probes derived from this fluorophore have unusually narrow emission 

 band width and have high absorbance and quantum yield. The prospects for preparation 

 of new DNA sequencing dyes with higher detectability and spectral resolution will be 

 presented. 



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