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

Chemical and enzymatic approaches were used to produce polynucleotide fragm ents containing acidlabile internucleotide P3 ' -N5 ' phosphoramidate bonds , either in a surface-bound form or in solution. The primer extension react ion utilizing 5 ' -amino-5 ' -deoxynucleoside 5 ' -triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces s imply by being premixed with the acidic matrices typically used for MALDI-T OF mass spectrometry of nucleic acids. This leads to detection procedures t hat are simple, robust and easy to automate. Utilizing this approach, a pol ymorphic site in the human ADRB3 gene was interrogated. Primer extensions w ith phosphoramidate analogs of dNTPs allowed for unambiguous discrimination of all possible genotypes.