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Limitations of the Different Methods for the Concentration Measurements of Human Genomic DNA and Oligonucleotide Samples

Published

Author(s)

Hua-Jun He, Erica V. Stein, Paul C. DeRose, Kenneth D. Cole

Abstract

We compared different methods to measure the concentrations of nucleic acids using samples of human genomic DNA (cell line origin), and a synthetic DNA oligonucleotide. NIST Standard Reference Material (SRM) 2082, a pathlength absorbance standard, was used to facilitate the absorbance measurements done with short pathlength cuvettes, microvolume spectrophotometers, and a microvolume plate reader. Control absorbance values were measured on a high accuracy dual-beam spectrophotometer and NIST calibrated pathlength cuvettes. Measurements of the human genomic DNA sample were also done with several types of fluorescent dye binding assays using different types of DNA calibrators. The human genomic DNA sample was characterized by using 6 different droplet digital PCR assays (amplicons located on different chromosomes) to measure the average copy number. The DNA copy numbers were converted to mass values using the molecular weight of the human genome. The results from the different methods (absorbance, fluorescent dye-binding, and the digital PCR) were compared and the caveats for each measurement method were discussed. The fluorescent dye binding methods gave different results with the human genomic DNA sample depending upon the type of DNA calibrator and the fluorescent dye that was used. The digital PCR assays results that were consistent with DNA concentration values determined from the absorbance values, but values obtained from digital PCR were sensitive to the droplet size and molecular weight of the human genome used in the calculations. NIST SRM 2082 can be used as a control or a pathlength calibrator to provide confidence in absorbance measurements. The components of SRM 2082, uracil and tryptophan, have spectrum that are similar to nucleic acids and proteins, respectively.
Citation
Biotechniques

Keywords

standard, measurements, absorbance, genomic DNA, human, digital PCR, fluorescent dye binding
Created February 1, 2018, Updated July 23, 2018