Quantification of an Oligonucleotide Containing a Sequence Failure Product: Comparison of Isotope Dilution Mass Spectrometry with other Quantification Methods

There is an increasing demand to develop a method for accurate quantification of DNA. Because current methods such as the ultraviolet (UV) absorption-based method are only capable of relative quantification, the quantification result depends completely on the reference material. To achieve accurate quantification of DNA, we have performed isotope dilution mass spectrometry (ID MS)-based quantification of oligonucleotides. We chose a 20-mer synthetic oligonucleotide as the analyte with a longer sequence failure product. Oligonucleotides sometimes contain sequence failure products, which are difficult to remove. It is important to quantify a target product in such a mixture. After evaluating the content of the sequence failure product, the analyte spiked with stable isotopically labeled deoxynucleotide monophosphates (dNMPs) was digested by enzyme to its constituent dNMPs or deoxynucleosides and quantified by liquid chromatography-mass spectrometry. The obtained mass fractions of the 20-mer oligonucleotide showed a good agreement with the results based on phosphate analysis by inductively-coupled plasma-optical emission spectrometry and ion chromatography. UV absorption, the general method for DNA quantification, resulted in under-estimation. On the other hand, the mass fraction obtained by the gravimetric method was over-estimated. This study shows that the ID MS method can determine the precise mass fraction of the target oligonucleotide with the sequence failure product and possesses potential as the primary method for the certification of DNA as a reference material.