Base Composition Analysis of Nucleosides Using HPLC

Alex Andrus1, Robert G. Kuimelis2

1 PE Applied Biosystems, Foster City, California, 2 Phylos, Inc., Lexington, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.6
DOI:  10.1002/0471142700.nc1006s01
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In this protocol, nuclease digestion of an oligonucleotide is followed by dephosphorylation and HPLC analysis of the monomers on a reversed‐phase C18 column. This method can be used to detect and quantitate a wide variety of nucleobase modifications in oligonucleotides. Integrated areas of the nucleoside chromatogram give precise quantitation of nucleoside composition when the relative extinction coefficient cofactors are applied to the sum of the areas of the four bases. The protocol is also useful for analysis of oligonucleotides containing conjugated moieties and carbohydrate modifications.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Sample Preparation, Digestion, HPLC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Sample Preparation, Digestion, HPLC

  Materials
  • 0.2 to 1.0 OD units (A 260) oligonucleotide sample
  • Master mix (see recipe)
  • 3 M sodium acetate
  • 95% ethanol
  • Acetonitrile/triethylammonium acetate buffer/water gradient (Table 10.6.2 and unit 10.5)
  • HPLC system (unit 10.5) with C‐18 reversed‐phase column
  • Additional reagents and equipment for reversed‐phase HPLC (unit 10.5.)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Andrus, A. 1992. Evaluating and Isolating Synthetic Oligodeoxynucleotides, Appendix 1. Applied Biosystems, Foster City, Calif. Available upon request.
   Eadie, J.S., McBride, L.J., Efcavitch, J.W., Hoff, L.B., and Cathcart, R. 1987. High‐performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition. Anal. Biochem. 165:442‐447.
   Krstulovic, A. 1987. Nucleic acids and related compounds. In In Handbook of Chromatography, Vol.1, Part A and B (G. Zweig and J. Sherma, eds.) pp. 161‐69 (A), 35‐64 (B). CRC Press, Boca Raton, Fla.
   Li, B. and Swann, P.F. 1989. Synthesis and characterization of oligodeoxynucleotides containing O6‐methyl‐,O6‐ethyl‐, and O6‐isopropylguanine. Biochemistry 28:5779‐5786.
   Singhal, R.P., Landes, P., Singhal, N.P., Brown, L.W., Anevski, P.J., and Toce, J.A. 1989. High‐performance liquid chromatography for trace analysis of DNA and kinetics of DNA modification. Biochromatography 4:78‐88.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library