IT SLICES, IT DICES, IT SEQUENCES DNA
   
   ORNL's portable ion trap is rapidly becoming the high-tech equivalent of
   the Veg-A-Matic. This versatile device, which is also used to sniff out
   explosives and analyze water and soil samples, is now part of a highly
   sensitive method of sequencing DNA.                 
   
   Scott McLuckey, Gary Glish, Gary VanBerkel, and Rose Ramsey, all of the
   Analytical Chemistry Division, use a technique called electrospray
   ionization to ionize DNA fragments for analysis in the ion trap. There
   they are subjected to multiple stages of mass spectrometry, which
   separates them according to mass and charge.               
   
   The electrospray process involves dissolving the DNA fragments in a
   solvent (usually water and then sending them through a high-voltage
   needle. At the end of the needle, the solution becomes electrostatically
   unstable, breaking into a fine mist or spray of charged droplets, known
   as an electrospray.       
   
   "Electrospray is an extremely soft ionization method," McLuckey says.
   "Historically, it has been very difficult to ionize DNA because it is
   very fragile. Most ionization methods require an energetic beam--a laser
   or an ion beam. This type of process usually transfers enough energy to
   the DNA's internal structure to cause it to break into pieces. We get
   around this problem by tricking the ion into entering the gas phase in
   the form of a charged droplet. When the solvent evaporates, a bare ion
   is left."    
   
   As these droplets dry off, their surface charge becomes very large and
   the ions are desorbed. The ions are then injected into the ion trap,
   which provides a mass spectrum. "From this spectrum, you can determine
   the molecular weight of the sample," says McLuckey. "It turns out that
   all combinations up to 14 bases have unique masses, so once we know the
   mass, we can determine the base composition--but not the order."
   
   The order of the bases is determined by selecting a DNA ion and
   energizing it until it breaks into pieces. By analyzing how the ion
   falls apart, the sequence of its bases can be determined. Repeating this
   process with other ions allows the entire DNA fragment to be
   sequenced.          
   
   Currently, this approach can handle sequences up to 12 bases long
   without any serious technical problems. "We're really a long way from
   sequencing sections of DNA as large as 500, but that's our goal," says
   McLuckey. "This research is less than a year old, so we're working our
   way up."     
   
   The value of this method lies not only in its potential as a sequencing
   tool but also in its ability to determine the precise composition of a
   sample. For example, some DNA bases are modified by methylation, a
   condition known to influence certain cell characteristics, although how
   this occurs is not well understood.                 
   
   "Other techniques used to sequence DNA typically cannot identify a
   modified base," says McLuckey. "Using mass spectrometry, the modified
   base stands out like a sore thumb.            
   
   "Down the road, the most valuable aspect of this technique may be its
   applications for locating and identifying modified DNA. I think it will
   probably be highly useful in cancer research once the genome is
   sequenced--once we know what's right, this will be very useful in
   identifying what's wrong.

   ------------------------------------------------------------------------
   
   Please send us your comments.
   
   Date Posted:  1/10/94  (ktb)