Comparison of three common DNA concentration measurement methods

Anal Biochem. 2014 Apr 15:451:18-24. doi: 10.1016/j.ab.2014.01.016. Epub 2014 Feb 1.

Abstract

Accurate measurement of DNA concentration is important for DNA-based biological applications. DNA concentration is usually determined by the ultraviolet (UV) absorption, fluorescence staining, and diphenylamine reaction methods. However, the best method for quality assurance of measurements is unknown. Here, we comprehensively compared these methods using different types of samples. We found that all three methods accurately determined the concentrations of high-purity DNA solutions. After digestion of DNA samples, concentration measurements revealed that the PicoGreen dye method was very sensitive to the degradation of DNA. The three methods displayed different anti-jamming ability when contaminants such as transfer RNA (tRNA), protein, and organic chemicals were included in DNA solutions. The diphenylamine reaction method gave the highest accuracy, with an average error of approximately 10% between measured and true values. The PicoGreen dye method was influenced by tRNA and protein, and the UV absorption method was susceptible to all kinds of impurities. Overall, the diphenylamine reaction method gave the most accurate results when DNA was mixed with contaminants, the PicoGreen dye method was most suitable for degraded DNA samples or DNA extracted from processed products, and the UV absorbance method was best for evaluating the impurities in DNA solutions.

Keywords: Comparison; DNA concentration measurement; Diphenylamine reaction; PicoGreen dye; UV absorption.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Plant / analysis*
  • DNA, Plant / chemistry
  • Diphenylamine / chemistry
  • Fluorescent Dyes / chemistry
  • Fluorometry*
  • Organic Chemicals / chemistry
  • Plant Leaves / genetics
  • Plants / genetics
  • Proteins / chemistry
  • RNA, Transfer / chemistry
  • Seeds / genetics
  • Spectrophotometry, Ultraviolet*

Substances

  • DNA, Plant
  • Fluorescent Dyes
  • Organic Chemicals
  • PicoGreen
  • Proteins
  • RNA, Transfer
  • Diphenylamine