Unit

UNIT 10.10 Sequencing Oligonucleotides by Enrichment of Coupling Failures Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

  1. David Alazard,
  2. James Russell

Published Online: 1 JAN 2006

DOI: 10.1002/0471142700.nc1010s23

Current Protocols in Nucleic Acid Chemistry

Current Protocols in Nucleic Acid Chemistry

How to Cite

Alazard, D. and Russell, J. 2006. Sequencing Oligonucleotides by Enrichment of Coupling Failures Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Current Protocols in Nucleic Acid Chemistry. 23:10.10:10.10.1–10.10.7.

Author Information

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

Publication History

  1. Published Online: 1 JAN 2006
  2. Published Print: DEC 2005

Abstract

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.

Keywords:

  • oligonucleotide sequencing;
  • MALDI-TOF mass spectrometry;
  • crude synthetic oligonucleotide;
  • coupling failures;
  • coupling efficiency;
  • solid-phase extraction