Molecular markers
DNA purification
Analyzing nucleic acid purity

Author: Rodney E. Shackelford, D.O., Ph.D. (see Authors page)

Revised: 29 May 2018, last major update January 2012

Copyright: (c) 2012-2018, PathologyOutlines.com, Inc.

PubMed Search: Analyzing nucleic acid purity

Cite this page: Shackelford, R.E. Analyzing nucleic acid purity. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/moleculardnapuranalyzingpurity.html. Accessed October 17th, 2018.
Definition / general
  • Many different methods are used to analyze nucleic acid purity; each has specific advantages and disadvantages
Spectrophotometric analysis (A260/280 method)
  • A260/280 method of determining nucleic acid purity is quick and inexpensive
  • Theory: nucleic acids absorb significant ultraviolet (UV) light at 260 nm, with the degree of ultraviolet light absorption being directly related to the nucleic acid concentration, by the Beer-Lambert law (Wikipedia: Beer-Lambert Law [Accessed 29 May 2018])
  • Extinction coefficient for single stranded DNA is 0.027 (ug/ml)-1 cm-1, for double stranded DNA is 0.020 (ug/ml)-1 cm-1, for single stranded RNA is 0.025 (ug/ml)-1 cm-1
  • Based on absorption at 260 nm, an optical density gradient or OD of 1.0 corresponds to 50 ug/ml for double stranded DNA
  • Proteins strongly absorb UV light at 280 nm, mainly due to the amino acids tyrosine, tryptophan, cysteine; thus, the concentration of contaminating proteins within a nucleic acid sample can be calculated from the absorbance ratios at 260 and 280 nm (A260/280)
  • A260/280: ~1.8 for pure DNA samples; ~2.0 for pure RNA samples
  • A260/280 falls with increasing protein contamination, is ~0.57 for pure proteins in solution without nucleic acids
  • Nucleic acid samples should be free of phenol, which absorbs strongly at 270 nm
  • Pure nucleic acids should have zero absorbance at 330 nm; absorption at 330 nm and above indicates visible light absorption by particulates in solution
Nucleic acid quantification with fluorescent dyes
  • Can quantify nucleic acids by measuring fluorescence intensity of dye bound nucleic acids
  • Useful to quantify relatively minute nucleic acid concentrations, helpful if sample has too many absorbing A260 contaminants
  • Accuracy due to specificity of nucleic acid binding and fluorescence
  • Method: DNA or RNA binding dye is added to a nucleic acid solution; solution is loaded into agarose gel or a surface like a plastic wrap
  • Nucleic acid samples of known concentration are added to unknown sample and nucleic acid concentration is estimated by comparing unknown to known samples
  • Although somewhat subjective, this method works well for samples with low concentrations of nucleic acids
  • This method can be made more exact using microplates or cuvettes measurements against a standard curve on a fluorescent photometer
  • Dyes include ethidium bromide, SybrGreen 1, cyanine dyes, RiboGreen, PcoGreen and DAPI