A STRATEGY FOR RAPID AND EFFICIENT DNA-SEQUENCING BY MASS-SPECTROMETRY

Two methods of solid-phase Sanger DNA sequencing followed by detection with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are demonstrated. In one method, sequencing ladders gener ated on an immobilized synthetic template were resolved up to the 63-m er including the primer Detection sensivity and resolution were suffic ient for sequence analysis in the given range, This approach is partic ularly suitable for comparative (diagnostic) DNA sequencing. A second method that has the potential for high throughput de novo DNA sequenci ng is also presented; it uses immobilized duplex probes with five-base single-stranded overhangs to capture an unknown DNA template serving as primers for Sanger DNA sequencing. The power of mass spectrometry i s demonstrated not only by its very high speed, but also by its abilit y to identify sequences that are not readable using gel electrophoresi s.