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Nucleic Acids Research, 2001, Vol. 29, No. 18 3864-3872
© 2001 Oxford University Press

Matrix-induced fragmentation of P3'-N5' phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms

Mikhail S. Shchepinov*, Mikhail F. Denissenko, Kevin J. Smylie, Ralf J. Wörl, A. Lorieta Leppin, Charles R. Cantor and Charles P. Rodi

Sequenom Inc., 3595 John Hopkins Court, San Diego, CA 92121, USA

Chemical and enzymatic approaches were used to produce polynucleotide fragments containing acid-labile internucleotide P3'-N5' phosphoramidate bonds, either in a surface-bound form or in solution. The primer extension reaction utilizing 5'-amino-5'-deoxynucleoside 5'-triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces simply by being premixed with the acidic matrices typically used for MALDI-TOF mass spectrometry of nucleic acids. This leads to detection procedures that are simple, robust and easy to automate. Utilizing this approach, a polymorphic site in the human ADRB3 gene was interrogated. Primer extensions with phosphoramidate analogs of dNTPs allowed for unambiguous discrimination of all possible genotypes.

* To whom correspondence should be addressed. Tel: +1 858 202 9073; Fax: +1 858 259 5735; Email: misha{at}bioch.ox.ac.uk


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