How it Works: Efficient Oligonucleotide Analysis and Design

Problem: Oligonucleotides are short sequences of nucleic acid that are often used as primers and probes for amplifying DNA in polymerase chain reactions (PCR), measuring gene expression levels in qPCR, or for detecting DNA or RNA in applications such as DNA microarrays, Southern blots, allele-specific oligonucleotide analysis, and fluorescent in situ hybridization (FISH). In order to effectively anneal to their target sequence, each manufactured oligo must be complementary to the target sequence without a tendency to form undesirable secondary structures. These secondary structures include hairpin formations and self-dimers, which render the probe unable to bind to the target sequence. In addition, properties such as GC content and melting temperature must also be closely monitored in order to optimize the efficiency of the application. Ensuring that all these parameters are considered can be a lengthy process and can occupy a significant quantity of laboratory time. These assessment and optimization measures need to be considered, but can hinder the streamlining of high-throughput protocols.

Solution: In order to address the need for a quick and easy-to-use method of oligo optimization, Integrated DNA Technologies (IDT) offers the SciTools suite of free, online oligo analysis and design tools. The SciTools suite provides a central resource where users can input any sequence and determine its properties. It is comprised of the OligoAnalyzer 3.1, a number of application-specific, pre-design and design tools, as well as a dilution calculator, resuspension calculator and mFold, which determines the folding properties of the oligo.

As the mainstay of the SciTools, the OligoAnalyzer enables quick and easy analysis of any oligo sequence through use of complex algorithms to assess various properties. In order to initiate the analysis, users simply enter their sequence, which can include standard and mixed bases, as well as DNA, RNA, methylated, locked, and phosphorothioated bases into the box provided. Once the sequence is entered, users can initiate a variety of different calculations.

By clicking on the ‘analyze’ icon, the OligoAnalyzer automatically calculates the physical properties of the sequence. These include length, GC content, melting temperature range, molecular weight, the extinction coefficient and the optical density (O.D), which are all displayed on-screen for quick and easy review. The sequence can subsequently be altered and reanalyzed, as required.

Clicking on the ‘hairpin’ icon directly links to the SciTools mFold function, which measures the folding properties of the sequence to determine the likelihood of a hairpin structure forming. Hairpin structures can prove highly problematic in a number of applications by hindering the ability of the oligo to anneal to its target sequence. The formation of self-dimers can have a similar effect. For example, a PCR primer that forms a self-dimer will decrease the concentration of free primer available for binding, thereby reducing both the efficiency and accuracy of the reaction. The potential to form primer-dimers can result in false positive data due to non-specific annealing. The probability for each type of non-specific binding is easily detectable by a simple click in the OligoAnalyzer.

As an additional level of functionality, clicking on the NCBI Blast icon initiates a comparison of the oligo sequence in the NCBI database and will calculate the statistical significance of the matches.

For further information and to explore the complete suite of SciTools, including the PrimeTime pre-design, RNAi design, and antisense design, visit www.idtdna.com.