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Utah Researchers Add Small-Amplicon Design Tool to Family of PCR Data Analysis Applications

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Researchers from the University of Utah's department of pathology have added one more tool to a family of applications that they've developed for analyzing data from high-resolution melting-based genotyping — a very sensitive method of identifying genetic mutations in PCR experiments by looking at differences in DNA melting point profiles.

Speaking with BioInform this week, Zachary Dwight, scientific software manager for Carl Wittwer's laboratory in Utah's pathology department, explained that the latest addition to the team's web-based suite, User Design, or uDesign, provides an automated method of designing small amplicons used in assays intended to clarify cases where the differences between the wild type and mutant sequences aren't clearly marked by their respective melting point curves.

For example, he said, the melting curve of a 100 base pair sequence rich in G's and C's with a single nucleotide variant might not be much different from a melting curve of the wild type. However, by reducing the length of the melting sequence to around 50 base pairs, for instance, the difference in melting temperatures of the two sequences should become clearer.

UDesign streamlines that short amplicon design process by automating the information search process, Dwight said. As explained in a poster presented at last month's Association for Molecular Pathology conference in Phoenix, uDesign takes care of "sequence retrieval, primer design, melting curve predictions for all products, and hairpin design if nearest-neighbor symmetry exists."

All users need to do is enter the appropriate reference SNP ID number into uDesign and the software's underlying algorithms do the rest, pulling relevant data from the National Center for Biotechnology Information's database, designing primers, assembling the amplicon, and then predicting the melting curves for the genotypes in question.

The AMP poster provides a little more detail about the results of uDesign's run. It states that the "melting curves are plotted and labeled for both genotypes and duplexes" and that it displays "melting temperatures of both primers and the product, GC content, the alleles present, and the frequency data retrieved from the NCBI." It also notes that prior to running the software, users have the option to do things like change default primer melting temperatures, move primers, or place constraints on primer length.

UDesign is part of a larger portfolio of web-based software that share similar algorithms for predicting and analyzing the melting point curves of PCR products. The software was developed in Wittwer's laboratory by Dwight, Wittwer, and a third colleague, Robert Palais, to support researchers running high-resolution melting experiments.

The current suite includes prediction tools such as uMelt, which was designed to predict the melting curves of PCR products; uMelt HETS, an extension of uMelt for predicting heteroduplex and heterozygote melting curves; and uAnalyze, which is used to compare predicted and experimentally determined melting points.

A detailed description of uMelt was published about two years ago in Bioinformatics. Last year, the developers published a paper describing uAnalyze in IEEE Transactions on Computational Biology and Bioinformatics, and a third paper describing uMelt HETS was just accepted for publication in Human Mutation.

Dwight, who is a co-author on the aforementioned papers, said that team has begun working along with an unnamed company on a new gene-scanning application. He could not go into details about what its capabilities will be but he says that it will likely be released next spring.

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