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

David Alazard1, James Russell1

1 Gen‐Probe, Inc., San Diego, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.10
DOI:  10.1002/0471142700.nc1010s23
Online Posting Date:  January, 2006
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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

     
 
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Table of Contents

  • Basic Protocol 1: Extraction, Desalting, and Concentration of Failure Sequences from a Crude Oligonucleotide
  • Basic Protocol 2: Preparation of Oligonucleotide Failure Sequences for MALDI‐TOF Analysis
  • Basic Protocol 3: MALDI‐TOF Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extraction, Desalting, and Concentration of Failure Sequences from a Crude Oligonucleotide

  Materials
  • Crude 5′‐O‐(4,4′‐dimethoxytrityl)‐protected (DMTr) oligonucleotide (1 to 2 mg from an 80‐ to 750‐µmol scale synthesis), ammoniacal solution
  • 0.1 and 2.0 M triethylammonium acetate (TEAA), pH 7.0, HPLC grade
  • Acetonitrile (MeCN), HPLC grade
  • Ultrapure (e.g., Milli‐Q) water
  • 17% or 18% (v/v) MeCN in 0.1 M TEAA, pH 7.0 (see recipe)
  • Vacuum centrifugation system (Speedvac)
  • Temperature‐controlled heating block
  • 5‐mL disposable plastic syringes with exposed Luer tips
  • C18 reversed‐phase oligonucleotide purification cartridges (e.g., Sep‐Pak Plus, Waters Corporation)

Basic Protocol 2: Preparation of Oligonucleotide Failure Sequences for MALDI‐TOF Analysis

  Materials
  • Concentrated oligonucleotide failure sequences (see protocol 1)
  • Ultrapure (e.g., Milli‐Q) water
  • 50 mg/mL ammonium citrate buffer, pH 9.4 (from Sequazyme Oligonucleotide Sequencing Kit, PerSeptive Biosystems, or see recipe)
  • 50 mg/mL 3‐hydroxypiconilic acid (3‐HPA) matrix (see recipe)
  • Ammonium‐activated cation‐exchange resin beads (see unit 10.1)
  • Parafilm
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Figures

Videos

Literature Cited

Literature Cited
   Alazard, D., Filipowsky, M., Raeside, J., Clarke, M., Majlessi, M., Russell, J., and Weisburg, W. 2002. Sequencing of production‐scale synthetic oligonucleotides by enriching for coupling failures using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Anal. Biochem. 301:57‐64.
   Isola, N.R., Allman, S.L., Golovlov, V.V., and Chen, C.H. 1999. Chemical cleavage sequencing of DNA using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Anal. Chem. 71:2266‐2269.
   Juhasz, P., Roskey, M.T., Smirnov, I.P., Haff, L.A., Vestal, M.L., and Martin, S.A. 1996. Applications of delayed extraction matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry to oligonucleotide analysis. Anal. Chem. 68:941‐946.
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   Limbach, P.A., McCloskey, J.A., and Crain, P.F. 1994. Enzymatic sequencing of oligonucleotides with electrospray mass spectrometry. Nucleic Acids Symp. Ser. 31:127‐128.
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   Pieles, U., Zürcher, W., Schär, M., and Moser, H.E. 1993. Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry: A powerful tool for the mass and sequence analysis of natural and modified oligonucleotides. Nucl. Acids Res. 21:3191‐3196.
   Polo, L.M., McCarley, T.D., and Limbach, P.A. 1997. Chemical sequencing of phosphorothioate oligonucleotides using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Anal. Chem. 69:1107‐1112.
   Roskey, M.T., Juhasz, P., Smirnov, I.P., Takach, E.J., Martin, S.A., and Haff, L.A. 1996. DNA sequencing by delayed extraction‐matrix‐assisted laser desorption/ionization time of flight mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 93:4724‐4729.
   Schuette, J.M., Pieles, U., Maleknia, S.D., Srivatsa, G.S., Cole, D.L., Moser, H.E., and Afeyan, N.B. 1995. Sequence analysis of phosphorothioate oligonucleotides via matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. J. Pharm. Biomed. Anal. 13:1195‐1203.
   Smirnov, I.P., Roskey, M.T., Juhasz, P., Takach, E.J., Martin, S.A., and Haff, L.A. 1996. Sequencing oligonucleotides by exonuclease digestion and delayed extraction matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Anal. Biochem. 238:19‐25.
   Taranenko, N.I., Allman, S.L., Golovlev, V.V., Taranenko, N.V., Isola, N.R., and Chen, C.H. 1998. Sequencing DNA using mass spectrometry for ladder detection. Nucl. Acids Res. 26:2488‐2490.
   Wu, H. and Aboleneen, H. 2000. Sequencing oligonucleotides with blocked termini using exonuclease digestion and electrospray mass spectrometry. Anal. Biochem. 287:126‐135.
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