Automated amplicon design suitable for analysis of DNA variants by melting techniques

Abstract

Background: The technological development of DNA analysis has had tremendous development in recent years, and the present deep sequencing techniques present unprecedented opportunities for detailed and high-throughput DNA variant detection. Although DNA sequencing has had an exponential decrease in cost per base pair analyzed, focused and target-specific methods are however still much in use for analysis of DNA variants. With increasing capacity in the analytical procedures, an equal demand in automated amplicon and primer design has emerged.

Results: We have constructed a web-based tool that is able to batch design DNA variant assay suitable for analysis by denaturing gel/capillary electrophoresis and high resolution melting. The tool is developed as a computational workflow that implements one of the most widely used primer design tools, followed by validation of primer specificity, as well as calculation and visualization of the melting properties of the resulting amplicon, with or without an artificial high melting domain attached. The tool will be useful for scientists applying DNA melting techniques in analysis of DNA variations. The tool is freely available at http://meltprimer.ous-research.no/ .

Conclusion: Herein, we demonstrate a novel tool with respect to covering the whole amplicon design workflow necessary for groups that use melting equilibrium techniques to separate DNA variants.

Fig. 1

Screenshot of the web interface for Variant Melting Profile (http://meltprimer.ous-research.no/). Single or batch mode is selected in the “run” drop down menu. Design history can be saved in an optionally registered user account

Fig. 2

Output results when entering genomic variants in batch mode

Fig. 3

Part of results output when designing primers for 657 rs numbers in the TP53 gene

Fig. 4

Screen capture of the result output. The melting profile of the amplicon is plotted as a function of base pair number in the amplicon. An artificial high temperature melting domain is extended onto one of the primers. The difference in melting between the two variants is shown. A zooming function allows for closer inspection of the melting profile. (https://hyperbrowser.uio.no/dev2/static/hyperbrowser/run_specific/036/36131/html/chart-0.html). The results table below summarizes the design, e.g. primers, fragment length, melting temperature and GC-clamp position

Fig. 5

Allele separation of an amplicon with primers selected, controlled and simulated by the Variant Melting Profile. Inserted is the melting profile. Alleles were separated by cycling the temperature from 52–49 °C, 20 times during capillary electrophoresis. Signal was recorded by laser-induced fluorescence

Fig. 6

HRM analysis of different sample with different genotypes in the polymorphic site identified by rs2252586

Fig. 7

HRM assay of twelve polymorphic sites. The differences between a known heterozyote sample and seven DNA samples tested for unknown genotypes in twelve different amplicons is plotted for each amplicon. The amplicon id is given as rs number. Please note that the scale of the x- and y-axis is different for each plot. Solid black line, homozygote 1 sample, dotted gray line, heterozygote sample, light gray line, homozygote 2 sample