Oligo Handling, Analysis, and Applications
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# Oligo quantification—getting it right

We occasionally get calls from researchers who take issue with the yield readings we supply, saying they differ from what the researcher has calculated after resuspension. This article highlights the importance of using the [right] molar extinction coefficient in calculations of oligonucleotide concentration.

Why include extinction coefficient in calculations?

Optical absorbance at 260 nm is routinely used to measure the concentration of nucleic acids present in a solution. Approximate conversion factors estimate that duplex DNA is about 50 μg/OD260, single-stranded RNA is approximately 40 μg/OD260, and single-stranded DNA is approximately 33 μg/OD260.

While these conversion factors may provide a reasonable estimate for long, essentially randomized sequences, they are less accurate for short oligonucleotides and repeating sequences. Conjugated double bonds are strong absorbers of light. They exhibit a property known as resonance, which causes them to absorb light of longer wavelengths (i.e., low energy). The molar absorptivity or extinction coefficient, and wavelength of maximum absorbance generally increase with increasing numbers of conjugated double bonds. Thus, the absorbance of each base is different, and base composition, sequence context, and sequence length all influence absorbance. In laymen’s terms, the extinction coefficient of a material describes the amount of light absorbed at a given concentration and distance travelled.

By how much does sequence composition affect extinction coefficient?

Would you believe that the extinction coefficient for a sequence as short as 6 bases can vary just by arranging the bases in a different order? Additionally, base composition can lead to significant differences in the extinction coefficient. See Table 1 for examples.

Greatest accuracy is therefore achieved when the exact value of ε260 is calculated for each oligo. Further, it is necessary to take into account the presence of oligo modifications, such as fluorescent dyes, which may have significant absorbance at 260 nm.

For these reasons, IDT calculates the extinction coefficient for every oligo synthesized using a nearest neighbor method. This value is then used to measure the yield for each oligonucleotide produced.

The nearest neighbor method

Nearest neighbor values for ε260 of dNTPs are:

Table 2. Nearest neighbor values for ε260 of dNTPs. Values are provided in L/mole·cm [1].

We then use the formula [2,3],

Absorbance is calculated using the Beer-Lambert Law:

A = log(Io/I) = ε × c × p

where, A is absorbance; Io and I are, respectively, the intensities of incident and transmitted light; c is the molar concentration of an oligonucleotide (mole/L); p is the length of the light path through the sample (cm); and ε is the molecule molar extinction coefficient (L/mole•cm).

Is there a program that can do that calculation for me?

The nearest neighbor calculation of extinction coefficient for any sequence can be calculated using the free IDT OligoAnalyzer® program (www.idtdna.com/SciTools), which will then go on to determine OD for that sequence.

Figure 1. 3 Easy steps for determining oligonucleotide extinction coefficient and concentration. The OligoAnalyzer® program can be accessed at www.idtdna.com/SciTools. Step 1: Paste or type in the oligo sequence of interest. Step 2: Click on the Analyze button. Step 3: Read out your results.

Know the limitations of your instrumentation. Most labs use inexpensive spectrophotometers that are focused on accessibility and ease of use. These advantages are important but sometimes come at the expense of measurement accuracy. Know the linear detection range of your instrument and stay within that range. Additionally, like all pieces of equipment, spectrophotometers may age or drift so it is important to monitor their performance over time. All IDT instruments are measured against NIST traceable standards [4] at regular intervals.

Thorough mixing is important.
For an accurate assessment of quantity it is essential that the material sampled be representative of the entire sample. Be sure to mix the sample to homogeneity to avoid under or over estimations of quantity. This is important not only during resuspension, but before all critical absorbance measurements. Concentration gradients may develop due to freeze/ thaw cycles, for example.

Volume delivery during sample preparation is important. While sample volume accuracy may not be critical for all instruments, it is for cuvette-based spectrophotometers. Generally speaking, the greater the transfer volume, the more accurate and precise transfer will be. So, it is best to use highest transfer volume possible. This is especially true for pipettes. Use them at or near their nominal volume; i.e., perform 20 μL transfers using a 20 μL pipette and not a 100 μL pipette. Even though both pipettes can make the transfer, the 20 μL pipette will transfer the volume more accurately and precisely.

References

1. Warshaw MM, and Tinoco I. (1966) Optical properties of sixteen dinucleoside phosphates. J Mol Biol, 20(1):29−38.
2. Cantor CR, Warshaw MM, Shipiro H. (1970) Oligonucleotide interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleolides. Biopolymers, 9(9):1059−1077.
3. Cavaluzzi MJ and Borer PN. (2004) Revised UV extinction coefficients for nucleoside–5‘–mono¬phosphates and unpaired DNA and RNA. Nucleic Acids Res, 32(1) e13.
4. http://www.nist.gov/traceability/

### Product focus—Oligo, modifications, dsDNA fragments

Custom Oligonucleotides and primers

You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.

Oligo modifications

Review a list of the common modifications IDT can add to oligonucleotides here. Not finding a modification you need on the IDT website? IDT will consider any modification you need. Just send your request to noncat@idtdna.com.

Custom dsDNA Fragments

Rather than annealing oligonucleotides to obtain dsDNA fragments, when your fragment size is 125 bp or longer, it might make more sense to order gBlocks® Gene Fragments. gBlocks Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.

Easy resuspension and dilution of oligonucleotidesScreen shots walk you through use of IDT free, online resuspension and dilution calculation tools. Learn the variety of units that can be used as input values.

Scale and yield—are they the same?—Understand the distinction between the amount of starting material vs the amount of final product recovered.

Getting enough full-length oligo?—The coupling efficiency achieved by an oligonucleotide manufacturer has a direct effect on the quality of the oligonucleotides produced. Find out why coupling efficiency should be important to you.

The Importance of Tm in Molecular Biology Applications—Predict and select the appropriate Tm for oligo hybridization steps (e.g., for PCR).

Browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Authors: Ellen Prediger, PhD is the Director of Scientific Communication and Thad Lane, BS is a Process Engineer at IDT.

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