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  • Title: Sequencing of production-scale synthetic oligonucleotides by enriching for coupling failures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
    Author: Alazard D, Filipowsky M, Raeside J, Clarke M, Majlessi M, Russell J, Weisburg W.
    Journal: Anal Biochem; 2002 Feb 01; 301(1):57-64. PubMed ID: 11811967.
    Abstract:
    A technique for sequencing oligonucleotides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is described. The series of coupling failure species are extracted from the dimethoxytrityl-on, full-length oligonucleotide in crude synthetic material using C18 stationary-phase cartridges. These concentrated failure species can be easily detected by MALDI-TOF, which determines the mass difference between spectral ions to identify a particular base. The solid-phase extraction step greatly enhances ion signals and mass resolution, and sequencing information is generally obtained from the 5' end up to the first three to four nucleotides at the 3' end. Complete sequence can be generated in conjunction with snake venom phosphodiesterase digestion of purified material. This method eliminates difficulties associated with other mass spectrometric sequencing techniques involving oligonucleotide length; structure; and sugar, base, and backbone modifications. Examples of sequencing a 17-mer composed primarily of 2'-O-methylribonucleotides and a single nonnucleosidic linker and a mixed sugar backbone 51-mer with 2'-O-methylribonucleotides and a homopolymer tail are reported in this study.
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