ANALYZING SEQUENCING REACTIONS FROM BACTERIOPHAGE-M13 BY MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY/

The current demand for improved DNA sequencing methodologies posed by the Human Genome Project has spurred the investigation of alternatives to gel electrophoresis. Matrix-assisted laser desorption/ionization ( MALDI) mass spectrometry has great potential for the rapid analysis of DNA fragments. Mock Sanger sequencing mixtures have been successfully analyzed by MALDI by pooling synthesized oligonucleotides correspondi ng to the M13 bacteriophage sequence. More recently, analyses of Sange r sequencing fragments enzymatically generated from synthetic template s of 45 or 50 bases were reported, In the present study, these feasibi lity demonstrations are extended to show MALDI sequencing from the M13 bacteriophage DNA template commonly used in actual Sanger sequencing. The results show sequence determination for extension products up to 35 bases in length, Different desalting and purification procedures we re investigated and it was found that salt could be efficiently reduce d by removal of the template in a post-reaction step. Work in progress to stabilize DNA by chemical modification, employed in conjunction wi th the methods described here, should enable significant extension of the length of readable sequence.