A strategy for establishing accurate quantitation standards of oligonucleotides: quantitation of phosphorus of DNA phosphodiester bonds using inductively coupled plasma-optical emission spectroscopy.
A novel approach for quantitation of DNA (oligonucleotides) with an unprecedented accuracy of approximately 1% is reported. Quantitation of DNA is commonly performed by measuring UV absorption or fluorescence from dyes intercalated into DNA. Both methods need accurate quantitation standards to yield more comparable results between laboratories. For establishing technically authentic standards for DNA quantitation, a new measurement approach carrying an inherent capability of absolute quantitation is demanded. The proposed approach is based on the stoichiometric existence of phosphorus (P) in DNA. The quantity of P from the phosphodiester backbone of a purified oligonucleotide was accurately determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) with yttrium internal standard via acid digestion. The number of moles of oligonucleotides was then calculated from that of P using the stoichiometry. The major issues regarding the validity of the suggested approach were (i) effective removal of extra P sources, (ii) quantitative recovery of P through the digestion process, and (iii) oligomeric purity of the target oligonucleotide. These issues were investigated experimentally using various analytical techniques such as matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis, electrical conductometry, UV spectrometry, and gravimetry. In conclusion, it is feasible to certify pure oligonucleotide reference materials with uncertainties less than 1% using the proposed approach.
Yang
Inchul
I
Division of Chemical Metrology and Materials Evaluation, Korea Research Institute of Standards and Science, 1 Doryong-dong, Yuseong-gu, Daejon 305-340, Republic of Korea.
Han
Myung-Sub
MS
Yim
Yong-Hyeon
YH
Hwang
Euijin
E
Park
Sang-Ryoul
SR
D004247:DNA / Q000032:analysis*; D009841:Oligonucleotides / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D010758:Phosphorus / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D013057:Spectrum Analysis
D009841:Oligonucleotides; D010758:Phosphorus; D004247:DNA