Cartridge Methods for Oligonucleotide Purification

Alex Andrus1, Robert G. Kuimelis2

1 PE Applied Biosystems, Foster, California, 2 Phylos, Inc., Lexington, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.7
DOI:  10.1002/0471142700.nc1007s01
Online Posting Date:  May, 2001
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Abstract

Protocols are given for purification of oligonucleotides by dimethoxytrityl‐sensitive and affinity desalting methods. The protocols are applicable for many of the convenient disposable products available for rapid oligonucleotide purification, clean‐up by selective adsorption, and elution on solid‐phase media. Many of these products are prepackaged, single‐use cartridges or columns filled with affinity or size‐exclusion media.

     
 
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Table of Contents

  • Basic Protocol 1: DMTr‐Selective Cartridge Purification
  • Basic Protocol 2: Affinity Cartridge Desalting of Detritylated Oligonucleotides
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: DMTr‐Selective Cartridge Purification

  Materials
  • 20% (v/v) and 100% acetonitrile
  • 2 M triethylammonium acetate (TEAA)
  • Tritylated oligonucleotide in ammonia deprotection solution
  • 3% (v/v) trifluoroacetic acid (TFA)
  • 1.5 M ammonium hydroxide
  • Oligonucleotide Purification Cartridges (OPC; PE Biosystems)
  • Vacuum manifold (Analytichem or National Scientific; optional) with an in‐line trap for waste and a water aspirator pump or small vacuum pump
  • 5‐mL disposable plastic syringes with exposed luer tips

Basic Protocol 2: Affinity Cartridge Desalting of Detritylated Oligonucleotides

  Materials
  • 0.1 M triethylammonium acetate (TEAA)
  • 50% (v/v) acetonitrile
  • Additional reagents and equipment for cartridge purification (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
   Applied Biosystems. 1991. New Applications for the Oligonucleotide Purification Cartridge. User Bulletin No. 59.
   Blocker, H., Frank, R., Heisterberg‐Moutsis, G., Kurth, G., and Meyerhans, A. 1991. Improved Process for the Purification of Synthetic Oligonucleotides. U.S. Patent No. 4,997,927.
   McBride, L., McCollum, C., Davidson, S., Efcavitch, J.W., Andrus, A., and Lombardi, S. 1988. A new reliable cartridge for the rapid purification of synthetic DNA. Biotechniques 6:362‐367.
   Mullah, B. and Andrus, A. 1996. Purification of 5′‐O‐trityl‐On oligoribonucleotides. Investigation of phosphate migration during purification and detritylation. Nucleosides Nucleotides 15:419‐430.
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