Quantitation of Oligonucleotides
The easiest way to determine the amount of an oligo present is by measuring the optical absorbance, OD260, of that oligo in solution. One OD260 unit is defined as the amount of oligonucleotide which, when dissolved in a volume of 1.0 ml, results in an absorbance of 1.0 when measured at 260 nm in a 1 cm path-length quartz cuvette. At IDT, each oligonucleotide we synthesize is individually quantitated by UV absorbance at 260 nm. Samples are assayed in duplicate and averaged. The mass amount of DNA is calculated from the measured OD value using the extinction coefficient, ε. The extinction coefficient is a physical constant that correlates OD260 with the amount of oligo present and is a value that is calculated uniquely for each sequence.
For more information about calculating an extinction coefficient.
For more information about calculating oligo quantity from OD260.
Unincorporated nucleotides and protecting groups can be present in a sample as undesired byproducts from oligo synthesis and can lead to inaccurate estimates of DNA mass. At IDT, all UV spectrophotometry is done on samples only after purification (desalted, PAGE, or HPLC). After quantitation, the oligo is lyophilized and shipped via overnight courier.
Estimates of DNA mass/concentration can also be obtained from samples run on acrylamide gels (PAGE) if compared to known standards. Best results will be obtained using denaturing urea-based gels (7M urea, 1x TBE, as are used in DNA sequencing). DNA bands can reliably be assessed by back-shadowing against a TLC plate using a hand held UV light source. The TLC plate will "glow" under UV stimulation; UV light absorbed by DNA bands in the gel will decrease the background fluorescence of the TLC plate and are seen as dark bands against a light background. Every oligonucleotide we make in 250 nm scale reactions or larger is examined this way as part of routine quality control at IDT. While reliable, backshadowing requires that relatively large amounts of DNA be used for visualization, around 5-6 mg. Greater sensitivity can be obtained using colorimetric stains or silver stains.
Estimates of DNA mass/concentration cannot be made for single stranded oligonucleotides using ethidium bromide staining with transmission fluorescence/excitation, as is commonly used with long double-stranded DNA samples. Ethidium binds nucleic acid by intercalation in regions of double-stranded structure. Single-stranded oligos can form regions of secondary structure that allow for ethidium binding; the availability of such sites varies considerably between oligos based on the amount of hairpin or dimer potential present. Thus, although single-stranded DNA can be detected by ethidium staining, mass estimation is inaccurate. Two oligo samples with identical mass/concentration but having a different sequence (and therefore different secondary structure and ethidium binding potential) can appear very different when visualized on a gel in this fashion. Agarose gels cannot be reliably used to quantitate short, single-stranded oligonucleotides.
Certain fluorescent dyes alter their fluorescent properties when bound to nucleic acids. Ethidium bromide, the best-known dye of this class, has moderate intrinsic fluorescence that increases 30-fold after binding DS DNA. Newer generation dyes have more favorable properties. For example, the cyanine dye PicoGreen (Molecular Probes) has very low intrinsic fluorescence that increases over a thousand-fold after binding DS DNA.
DNA-binding dyes usually display a preference for DS DNA versus SS DNA. For example, ethidium bromide and propidium iodide preferentially bind DS DNA (binding via intercalation). The Hoechst dyes and DAPI also preferentially bind DS DNA (minor-grove binders). GelStar Nulceic Acid Stain (BioWhittaker) can be used to visualize both SS and DS DNA in gels. OliGreen and SYBR Green II (Molecular Probes) preferentially bind SS DNA. OliGreen can be used to quantitatively assay oligonucleotides in solution and SYBR Green II can be used to quantitatively assay oligonucleotides in gels. Unlike OD260 measurements, the resulting fluorescence signal is not directly quantitative, although accurate estimates can be made by comparison to a standard curve. These methods are extremely sensitive, having detection limits approximately 10,000-fold lower than a direct optical absorbance assay.
Mark Behlke M.D., Ph.D.
Vice President, Molecular Genetics and Bioinformatics
Integrated DNA Technologies | 
