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Analyzing Sequencing Reactions from Bacteriophage M13 by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry
Author(s) -
Mouradian Stéphane,
Rank David R.,
Smith Lloyd M.
Publication year - 1996
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(199609)10:12<1475::aid-rcm696>3.0.co;2-c
Subject(s) - sanger sequencing , chemistry , mass spectrometry , oligonucleotide , dna sequencing , matrix assisted laser desorption/ionization , dna , combinatorial chemistry , chromatography , computational biology , desorption , biochemistry , organic chemistry , biology , adsorption
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 corresponding to the M13 bacteriophage sequence. More recently, analyses of Sanger sequencing fragments enzymatically generated from synthetic templates of 45 or 50 bases were reported. In the present study, these feasibility 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 were investigated and it was found that salt could be efficiently reduced by removal of the template in a post‐reaction step. Work in progress to stabilize DNA by chemical modification, employed in conjunction with the methods described here, should enable significant extension of the length of readable sequence.