Accurate nucleic acid concentrations by nuclear magnetic resonance

M. J. Cavaluzzi, D. J. Kerwood, P. N. Borer

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Determination of the concentration of biochemical samples often yields values with uncertainties of 10-20% or more. This paper details a protocol for use with 500- to 600-MHz NMR spectrometers to measure ∼1 mM concentrations within ± 1-3% accuracy. With suitable precautions, all compounds have equal NMR "absorption coefficients" for protons. About 2 mg of sample are needed for proteins and nucleic acids with MW = 5000, although less accurate determinations could be made with smaller amounts. The technique utilizes standardized internal reference reagent compounds, cacodylic acid or 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt. Spectra were signal-averaged using long interpulse delays so that integrals of nonexchangeable protons could be quantified relative to the reference standard. Accurate determinations require careful optimization of the homogeneity of the magnetic field and meticulous attention to sample preparation and spectral processing. The main source of error is usually the accuracy of micropipets. If sample preparation errors could be eliminated, the lower limit of accuracy with the current generation of NMR spectrometers is probably near 0.4%. However, this would require >99.5% sample purity. Methods are described to establish the concentration of the standards, and applications are illustrated with DNA mono- and oligonucleotides. Similar procedures should apply to proteins, polysaccharides, and other biomolecules, with about the same accuracy and precision.

Original languageEnglish (US)
Pages (from-to)373-380
Number of pages8
JournalAnalytical Biochemistry
Volume308
Issue number2
DOIs
StatePublished - Oct 15 2002

Fingerprint

Nucleic Acids
Protons
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Cacodylic Acid
Spectrometers
Magnetic Fields
Oligonucleotides
Uncertainty
Polysaccharides
Proteins
Research Design
Salts
Sodium
Biomolecules
Acids
DNA
Magnetic fields
Processing

Keywords

  • Concentration
  • DNA
  • Extinction
  • NMR
  • Protein
  • Quantitative
  • Reference
  • RNA

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Accurate nucleic acid concentrations by nuclear magnetic resonance. / Cavaluzzi, M. J.; Kerwood, D. J.; Borer, P. N.

In: Analytical Biochemistry, Vol. 308, No. 2, 15.10.2002, p. 373-380.

Research output: Contribution to journalArticle

Cavaluzzi, M. J. ; Kerwood, D. J. ; Borer, P. N. / Accurate nucleic acid concentrations by nuclear magnetic resonance. In: Analytical Biochemistry. 2002 ; Vol. 308, No. 2. pp. 373-380.
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