LUMINESCENT LABELING FOR DNA SEQUENCING
   
   The need for labeling and detection methods that could be easily
   automated to greatly increase the rate of sequencing DNA fragments is
   the motivating factor behind the luminescent labeling system being
   developed by Gilbert M. Brown of the Chemistry Division. The system uses
   the lanthanide ions samarium(III), europium(III), terbium(III), and
   dysprosium(III), bound by a chelating agent, as labels for the four DNA
   bases.       
   
   Luminescence from these rare-earth ions is generated by exciting the
   naphthalene group attached to the chelating agent. "This is an energy
   transfer process," says Brown. "We shine a light on the naphthalene,
   which has a long-lived excited state. It eventually gives up this
   excitation energy to the lanthanide ion, which responds by emitting
   light." Because of the way the lanthanide ions are linked to
   naphthalene, a single wavelength of light can excite all four labels,
   each of which emits light of a characteristic wavelength.                  
  
   
   DNA fragments are conventionally labeled with radionuclides, such as
   sulfur-35 or phosphorus-32. The labeled fragments are then separated by
   electrophoresis, and the position of each fragment is detected by
   autoradiography. This approach provides high sensitivity for detection
   but also presents problems, including the potential for radiation
   exposure, high disposal costs, and limited shelf lives of radionuclides.
   Using lanthanide ions eliminates these problems.                 
   
   Lanthanide ions offer several advantages over the fluorescent organic
   dyes currently in use. The fluorescent dyes have emission bandwidths
   that overlap, sometimes making it difficult to distinguish between
   bases. The emission bandwidths of the lanthanide ions are narrow, even
   at room temperature in fluid solution, allowing them to be detected
   simultaneously with minimum overlap.                
   
   "We are interested in designing a labeling system in which multiple
   labels could be detected simultaneously," Brown says. "From the sharp
   nature of the emission spectra, these elements appear ideal for
   detection."         
   
   Because the lifetimes of the excited states of these ions are relatively
   long, emission detection can be time-gated, virtually eliminating
   signals from background sources. "In time-gated detection, a pulsed
   excitation source allows a time delay between excitation and detection,"
   says Brown. "After excitation, we delay turning on the detector until
   sources of interfering light such as scattered excitation light, Raman
   scattering, and impurity fluorescence have died down, allowing us to
   identify bases with greater accuracy."              
   
   Another advantage of this system is that it is compatible with both
   capillary gel electrophoresis, which is considerably faster than
   conventional sequencing using slab gel electrophoresis, and computer
   collection and analysis of sequence data. "We'll be able to use a
   computer to look at the luminescence of the labeled DNA as it goes by,"
   says Brown. These factors, combined with the certainty provided by the
   sharp spectral emission lines of the lanthanide ions, should provide a
   sequencing rate that is considerably higher than that achieved using
   conventional methods.            
   
   "This is the poor man's way to increase sequencing rates," Brown says.
   "It's not the fastest method around, but for researchers who don't have
   a half million dollars' worth of equipment, it ought to be a big
   improvement.
   

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   Date Posted:  1/10/94  (ktb)