Determining the physical characteristics of your oligos

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 (T_m), molecular weight, extinction coefficient, and optical density. Access the OligoAnalyzer Tool from the “Tools” menu on top of any page on the IDT website (Figure 1).

Figure 1. Select the OligoAnalyzer Tool from the TOOLS menu on any IDT webpage. The OligoAnalyzer Tool link is found under the 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 T_m, it is important to enter the Mg⁺⁺ and dNTP concentrations you will use in your experiment (Figure 2, arrow and box C). Note that the T_m reported on the specification sheet shipped with IDT oligos uses default settings of 0 nM Mg⁺⁺ and 0 mM dNTPs, so will undoubtedly differ from the T_m calculated here. When you press “Analyze” (Figure 2, arrow D) the tool will produce the complementary sequence, GC content, T_m, molecular weight, and extinction coefficient for you. Additionally, you can run Self-Dimer and Hairpin analyses of your sequence from the Sequence entry screen (below Figure 2, arrow D).

Entry page on the OligoAnalyzer Tool. Insert your oligo sequence in the box (A), which accommodates DNA, RNA, mixed bases, locked nucleic acids, and a variety of other modifications (B). After adjusting the concentration of Mg⁺⁺ and dNTPs, or selecting the preset qPCR parameter from the dropdown (C), press Analyze (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 T_m should be lower than the temperature at which the oligo will be used (Figure 3).

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 T_m should be lower than the temperature at which the oligo will be used.

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

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 learn more. And, of course, you can always contact an IDT Customer Care Representative.