Design Software For Oligos, Primers, And Probes

Technological improvements in computing also make choosing software difficult—even for the software designers. For example, Wojciech Rychlik, president of Molecular Biology Insights, says that one of the challenges in creating probe design software is dealing with the complications brought about by ever-changing operating systems. “The entire field of sequencing is changing very rapidly,” agrees Bob Steinhauser, director of marketing at DNASTAR. “With these changes come changes in data files and changes in the applications that users want to have available to them. Updates are constantly being made in the software to keep up with these needs.”

Many other factors collude to make the road to probe design software a bumpy one, including the need to account for a multitude of factors simultaneously. “Software design needs to account for model accuracy, model tractability, user interface and usability, and reproducibility,” says Andrew Peek, director of bioinformatics at Integrated DNA Technologies (IDT), who singles out model accuracy as “probably the most important factor in scientific software.” Kay Brown, vice president of business development at Premier Biosoft, agrees that “the hardest part of designing this software is the complexity in balancing multiple criteria for overall assay success. The sophisticated search algorithm calculates all properties of every possible primer and probe within the allowed length and tolerance values and they must meet all specified criteria. It is rejected if any parameter falls outside of its tolerance limits.”

IDT’s philosophy includes allowing scientists as much, or as little, control over the design process as they desire. “We allow the user to control a large number of parameters in the design process,” explains Peek, “so while you might be able to treat the designs as a ‘black-box’ if you don’t want to get involved in the details, we allow the user to open the box and delve deeper into the underlying computational models, if they so choose.” IDT also offers custom design services.

Among IDT’s free software tools are OligoAnalyzer, which models several properties of oligos to analyze melting temperature, absorbance, molecular weight, and thermodynamics properties, and PrimerQuest, which designs primers for standard PCR, and primers and probes for qPCR. In addition, Peek adds, “IDT provides applications for the design of siRNAs and antisense sequences for gene expression knockdown, microarray probe design for gene expression studies and for designing [single nucleotide polymorphism] discriminating, or [locked nucleic acid] containing, probes.”

Brown claims that Premier Biosoft’s Beacon Designer is the best selling qPCR design software on the market, with more than 2,000 labs around the world using it. An advantage of this software is its flexibility—it supports all major qPCR assays, including TaqMan, Beacon, SYBR Green, FRET, NASBA, Scorpions, and LNA. Bio-Rad and Stratagene now offer Beacon Designer with their qPCR thermal cyclers. “Two unique features [of the software] that set it apart from competitive products are Avoid Cross Homology and Template Structure Search,” says Brown. “The primers and probes are automatically designed avoiding regions of homology and template structures identified, making the assays highly specific and efficient.”

Premier Biosoft also offers a program called AlleleID, which helps to address the challenges of pathogen detection, bacterial identification, or taxa/species discrimination using microarrays and qPCR assays. AlleleID can design species-specific, or even cross-species, probes for microarrays or TaqMan, TaqMan MGB, SYBR Green, and molecular beacon qPCR assays. Lastly, Premier Biosoft also offers Xpression Primer, which designs tagged primers for expression cloning with systems such as Gateway, BD In-Fusion, epitope, and TOPO tools. You can also use it with other expression systems.

Premier Biosoft is particularly concerned with involving customers in product improvements. “We handle every suggestion made by every user with the utmost care,” says Brown. “We have created a mechanism for gathering, documenting and responding to all suggestions and feature requests. We respond by screening and analyzing each suggestion or requirement and implementing it in the product. We release new versions with enhancements about every 3-4 months.”

Roche Applied Science also offers several design tools to suit varied interests. Their ProbeFinder software works with their online Assay Design Center and their Universal Probe Library. “The software in seconds calculates and ranks which probes of the Universal Probe Library are compatible with the sequence of interest,” says Rob van Miltenburg, global marketing director for qPCR and NAP systems for Roche Applied Science. “Comprehensive information about target sequence, primer sequences, intron-exon boundaries and alternative assays for the same sequence of interest are reported. The combination of ProbeFinder software with the 165 probes of the Universal Probe Library offers the customer overnight access to gene expression assays for more than 10 different species.” For design software for SYBR green assays, van Miltenburg recommends their Primer 3.0 or Oligo 5.0 programs. Roche also

These are but some of the current software offerings to consider. Also available are: Bio-Rad’s WinMelt and MacMelt, software for designing primers in mutation detection, Applied Biosystems’ Primer Express, an oligo design program for use with their ABI PRISM 7900HT and 7000 Sequence Detection Systems, and Sequenom’s MassARRAY QGE, which designs primers for multiplex assays. Another choice is Molecular Biology Insights’ Oligo, primer analysis software that “performs a range of functions for researchers in PCR and related technologies—consensus, multiplex and degenerate primers, oligonucleotide database, secondary structure, and more,” says Rychlik. Oligo has been an industry leader in oligo analysis software since 1989, according to Rychlik. “We're planning to release a major upgrade soon: Oligo 7 has expanded functionality, including qPCR, molecular beacons, siRNA probes, and batch file processing,” he adds.

As you peruse the range of features in the various probe design software available, consider also what effect future technological developments might have on your choice. It is, of course, impossible to predict this, but Peek believes that design software will become more complex: “As genetic design and engineering becomes an off the shelf commodity, any kind of design software will be both increasingly needed and then increasingly complex. Increasing need will be for the more mundane tasks of experimental design, where an individual researcher cannot perform the large number of computational tasks in a manual fashion. Increasing complexity will result from the movement and modeling of the biophysical properties of small nucleic acids, in a simple salt solution with one or two enzymes, to modeling of entire biological systems.” But with the frequent software updates released by most companies, you will likely stay up to speed no matter which software you choose.