SEQUENCING BY FOURIER TRANSFORM MASS SPECTROMETRY                
   
   Another variation on the theme of sequencing by mass spectrometry is
   offered by Bob Hettich and Michelle Buchanan, both of the Analytical
   Chemistry Division. While most approaches to sequencing DNA using mass
   spectrometry focus on measuring, molecular masses, Hettich and Buchanan
   are applying Fourier transform mass spectrometry (FTMS) to investigating
   the structure of modified DNA segments. The ion-trapping and
   manipulation capabilities of FTMS combined with its highly accurate
   measurement capabilities suggest that this technique has tremendous
   potential for investigating the structure of organic molecules.            
         
   
   The fragile nature of DNA molecules makes them difficult to study using
   conventional mass spectrometry. Hettich and Buchanan are avoiding this
   problem by using a technique known as matrix-assisted laser desorption,
   originally developed for examining proteins and peptides. For this
   method, the DNA segments are first mixed with a matrix compound
   (nicotinic acid) and then dried onto the tip of a stainless steel probe.
   The mixture is then desorbed with a 266-nanometer beam from a Nd:YAG
   (neodymium: yttrium aluminum garnet) pulsed laser.                      
   
   Because the nicotinic acid strongly absorbs electromagnetic radiation at
   266 nanometer, it serves as a buffer between the laser and the DNA,
   allowing the DNA segments to be carried into the gas phase with minimal
   fragmentation. The resulting DNA ions are then trapped in the FTMS cell
   where they can be studied in detail to determine their molecular weights
   and sequences.
   
   Once the molecular weights of the ions have been determined, they can be
   selectively fragmented by filling the FTMS cell with argon gas and
   studying the resulting collisions between the ions and the argon
   molecules. The fragmented ions provide detailed information about the
   sequence and structure of the original DNA segment. This approach has
   been used successfully to determine the sequence of segments up to six
   bases long. Improvement of the process should allow detection and
   characterization of even larger segments.                  
   
   In addition to determining the order of bases in each DNA segment, this
   process also determines the types and locations of chemical
   modifications of the DNA, such as the addition of alkyl groups to the
   DNA bases. "The main thrust of studying modified bases is to understand
   the molecular changes that occur in DNA as a result of cancer or
   mutation," Hettich says.         
   
   Currently, research is focused on two specific tasks. First, Hettich and
   Buchanan want to optimize the matrix-assisted laser desorption technique
   to allow investigation of larger DNA segments. "Nicotinic acid absorbs
   energy well at 266 nanometers," says Hettich, "but, unfortunately, so
   does DNA. The trick is to find another suitable laser matrix that
   absorbs energy well at a wavelength at which DNA does not. This should
   allow us to desorb larger pieces of DNA without them breaking up."         
               
   
   Secondly, they want to extend the instrumental capability of the FTMS to
   detect and examine larger DNA ions. "Developments in matrix-assisted
   laser desorption FTMS," Hettich says, "should help determine the
   potential of this technique for the measurement and structural
   characterization of large DNA fragments required for DNA sequencing.
   

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   Date Posted:  1/10/94  (ktb)