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Background  DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several  Naegleria  and  Cryptosporidium  amplicons generated  in vitro  with  in silico  DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria.    Results  The SYTO9 melting curve profiles of three species of  Naegleria  and two species of  Cryptosporidium  were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated  in vitro . Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts  in vitro .    Conclusion  Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for different species of  Naegleria  and  Cryptosporidium  and the complex melting profiles generated  in vitro  using SYTO9 verified that the complex melting profile of PCR amplicons was solely the result of DNA dissociation. Other data outputs from these simulations were also used to identify the melting domains that contributed to the observed melting peaks for each of the different PCR amplicons.

bmc bioinformatics

Use of DNA melting simulation software for in silico diagnostic assay design: targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes