AlleleID From Premier Biosoft

The ability to monitor the progress of PCR in real-time has revolutionized PCR-based quantification of DNA and RNA. The technique, known as real-time or quantitative PCR (qPCR), now plays a key role in scientific research. My group purchased AlleleID, a program for designing qPCR and microarray oligos, a couple of months ago. I have been using it frequently and am happy with it.

AlleleID designs species and taxa identification assays, for both qPCR (TaqMan®, TaqMan® MGB, SYBR® Green and molecular beacon assays) and microarray experiments. I can load sequences with homologies as high as 99% and can rely on AlleleID to find species- and taxa- specific primers or probes. AlleleID is the only tool I am aware of that can handle such oligo design challenges.

My PhD project aims at characterizing the functional diversity among 5 arbuscular mycorrhizal fungi (AMF) species (Glomus intraradices, Glomus claroideum, Glomus mosseae, Gigaspora margarita, Scutellospora pellucida) in symbiosis with Medicago truncatula. The molecular work of this project depends on specific and quantitative detection of those 5 AMF species when they have co-colonized the roots of Medicago t.

In more detail, my goal was to design 5 SYBR® Green primer pairs that would amplify each of the species uniquely, without amplifying the rest of the four. I had already aligned all the subspecies/strain sequences for each species before I discovered AlleleID. I, therefore, started by loading these alignment files, instead of using its built-in Clustal W algorithm.

To design a single SYBR® Green primer pair for unique identification of the first species, I made use of AlleleID's convenient 'bind to' and 'avoid' boxes. They tell the program which sequences I am interested in, and which must be strictly avoided. AlleleID was able to design a single primer pair that would amplify only the species of interest and at the same time allow the amplification of the different strains (or sub-species) of the species in question. The primer pair was located within region conserved across the sub-species of my species of interest.

I repeated the process for the remaining four species. For certain species, primer pair design restricted to the conserved region was not possible. I then chose AlleleID's “Mismatch Tolerant” primer design option, in which I entered the uppermost limit for allowed mismatched bases. With this selection, AlleleID designed the best possible oligos that contained the fewest possible mismatched bases while meeting all other design parameters. AlleleID not only minimized the total number of mismatched base, but also located them as close as possible to the 5' end of the oligo, to maximize the probability that it would bind to all of the sub-species.

To ensure specificity, it is possible to use AlleleID's BLAST search capabilities from within the program. AlleleID can not only show you the BLAST results, but can also interpret them automatically to locate the primers and probes to avoid false priming when necessary. While working with AlleleID, I experimented with its "Minimal Set" functionality. It is one of the most innovative features in the program. It helps design the fewest number of primers and probes that uniquely identify each of the desired species/strain/taxa from the mix, saving reagent costs. For taxa or cross species assays, this feature is especially useful when the group or taxa are highly dissimilar.

PREMIER Biosoft seems to have selected the default parameters with considerable thought. I found that the default values almost always succeeded. Just load the sequences, select options, press the Search button, and you have a fairly good assay design. For especially challenging situations, the default values sometimes fail. The status line then offers insights into what values may have caused the failure. I experimented with relaxing those parameters and, in a few tries, was able to design the assay successfully in most cases.

If you already have successfully designed primers and/or probes, AlleleID offers the capability to accommodate your proven designs. Using its “Evaluate” function, you can complete the design task by taking advantage of the algorithmic prowess of the program.

AlleleID is an especially useful tool for designing qPCR diagnostic assays and microarrays for rapid detection of pathogens or bacteria. Applications can be as diverse as environmental monitoring, prevention/control of infectious diseases, development of surveillance tests or biodiversity studies. The latest version of the program includes support for splice variant arrays to detect gene splicing and alternative splicing events. I did not play with this functionality.

The ease of using AlleleID is due partly to the streaming multimedia tutorial available on PREMIER Biosoft's website. It explains the search concepts used by the program quite well and is an effective tool to get started. I was able apply the program to my design goals effectively in just a few minutes of working with it after viewing the tutorial. The program is backed up by a strong technical support team. Support staff helped me whenever I had technical questions. They also provided knowledgeable solutions and suggestions to my problems. I own a windows version of AlleleID which I can run on Windows ME, NT 4.0, 2000 and XP Professional. A native OS X version is also available.