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Nucleic Acids Research, 2001, Vol. 29, No. 21 e104
© 2001 Oxford University Press

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

John R. Edwards1,2, Yasuhiro Itagaki3 and Jingyue Ju1,2,*

1Laboratory of DNA Sequencing and Chemical Biology, Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA, 2Department of Chemical Engineering and 3Department of Chemistry, Columbia University, New York, NY 10027, USA

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3'-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.

* To whom correspondence should be addressed at: Room 405A, Russ Berrie Medical Science Pavilion, Columbia University College of Physicians and Surgeons, 1150 St Nicholas Avenue, New York, NY 10032, USA. Tel: +1 212 304 7369; Fax: +1 212 304 5515; Email: dj222{at}columbia.edu


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