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Curr Protoc Nucleic Acid Chem. 2006 Jan;Chapter 10:Unit 10.10. doi: 10.1002/0471142700.nc1010s23.

Sequencing oligonucleotides by enrichment of coupling failures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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  • 1Gen-Probe, Inc., San Diego, California, USA.


A technique for sequencing oligonucleotides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is described. Coupling failures are extracted from the full-length 5'-O-DMTr-oligonucleotide in a crude synthesis using C18 purification cartridges. Enhanced signal and resolution of the failure ions are demonstrated during MALDI-TOF analysis. Sequencing information is confirmed by the mass difference between coupling failures to identify a particular base or structural modification. This procedure eliminates difficulties associated with other mass spectrometric techniques, such as interpretation of data, oligonucleotide length, and backbone, sugar or terminal alterations. An example of sequencing a 16-mer composed of deoxyribonucleotides, 2'-O-methylribonucleotides, and a non-nucleosidic linker is reported.

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