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Determining the physical characteristics of your oligos

The OligoAnalyzer™ Tool

Use the OligoAnalyzer Tool to determine many of the physical characteristics of your oligonucleotides. By simply inputting your sequence, you can find out its length, GC content, melting temperature range, molecular weight, extinction coefficient, and optical density.

IDT offers a powerful analytical tool for determining the properties of oligo sequences—the OligoAnalyzer Tool. This tool calculates the physical characteristics of any oligo sequence, including length, GC content, melting temperature (Tm), molecular weight, extinction coefficient, and optical density. Access the OligoAnalyzer Tool from under the “Tools” heading on any page of the IDT website (Figure 1).

Use the IDT tools menu to access OligoAnalyzer
Select the OligoAnalyzer Tool from the TOOLS heading on any page of the IDT website. The OligoAnalyzer Tool link is found under Oligo Design & Handling section. 

Enter your sequence into the “Sequence” box in the 5’ to 3’ orientation (Figure 2, arrow A). The OligoAnalyzer Tool accommodates DNA, RNA, mixed bases, and a variety of modifications, which can be selected from the menus under the Sequence box (Figure 2B). To get the correct Tm, it is important to enter the Mg++ and dNTP concentrations you will use in your experiment (Figure 2, arrow and box C). Please note that the Tm reported on the specification sheet shipped with IDT oligos uses default settings of 0 nM Mg++ and 0 mM dNTPs, and thus, will usually differ from the Tm calculated here. The “Analyze” button (Figure 2, arrow D) will produce the complementary sequence, GC content, Tm, molecular weight, and extinction coefficient. You can also run Self-Dimer and Hairpin analyses of your sequence from the Sequence entry screen (below Figure 2, arrow D).

Adjust experimental conditions to analyze Tm, GC content, molecular weight.
Entry page of OligoAnalyzer Tool. Insert your oligo sequence in the box (A), which will accommodate DNA, RNA, mixed bases, locked nucleic acids, and a variety of other modifications (B). After adjusting the concentration of Mg++ and dNTPs or choosing the preset qPCR parameter from the dropdown (C), select the analyze button (D). 

The Gibbs free energy (ΔG) value gives an indication of the strength of the secondary structure. IDT recommends the ΔG value be more than –9 for self-dimers and hetero-dimers. For hairpins, the Tm should be lower than the temperature at which the oligo will be used (Figure 3).

Primer self-dimer and hairpin analyses using OligoAnalyzer.
Run self-dimer and hairpin analyses. Review the calculated ΔG values for the different self-dimer permutations. The value for the most stable formation should have a ΔG value greater than –9 for self-dimers and hetero-dimers (not shown). For hairpins, the Tm should be lower than the temperature at which the oligo will be used.

If you need help with the program, the “Instructions” link provides step-by-step guidance on how to use the OligoAnalyzer Tool. The “Definitions” link gives an explanation of terms and equations the program uses to determine Tm and extinction coefficient (Figure 4).

Instructions and definitions links for OligoAnalyzer
Online resources to help you with the OligoAnalyzer program. Use either the instructions or definitions links above the Analyze button to learn more about this tool. 

Go to our login page to watch our video tutorial to learn more. And, of course, you can always contact an IDT Customer Care Representative.


Published Jun 15, 2012
Revised/updated Nov 25, 2020