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

ABSTRACT: 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.

SUBMITTER: Shchepinov MS

PROVIDER: S-EPMC55906 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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

Shchepinov M S MS Denissenko M F MF Smylie K J KJ Wörl R J RJ Leppin A L AL Cantor C R CR Rodi C P CP

Nucleic acids research 20010901 18

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 detec ...[more]

PMID: 11557819

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