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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.

Rasmussen JP, Saint CP, Monis PT - BMC Bioinformatics (2007)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cooperative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Salisbury, SA, Australia. paul.rasmussen@sawater.com.au

ABSTRACT

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.

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Comparison of melting profiles obtained using different intercalating dyes and purified Naegleria amplicons. First derivative plots for purified amplified DNA from Naegleria fowleri (red line), Naegleria lovaniensis (blue line) and Naegleria australiensis (black line) melted in the presence of either SYTO9 (A), SYBR Green (B), LC Green I (C) or SYTO59 (D).
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Figure 3: Comparison of melting profiles obtained using different intercalating dyes and purified Naegleria amplicons. First derivative plots for purified amplified DNA from Naegleria fowleri (red line), Naegleria lovaniensis (blue line) and Naegleria australiensis (black line) melted in the presence of either SYTO9 (A), SYBR Green (B), LC Green I (C) or SYTO59 (D).

Mentions: To establish that the observed similarity in predicted and actual meting curves was not a "dye-specific" phenomenon, N. australiensis, N. fowleri and N. lovaniensis amplicons were purified and added in equal amounts (ng) to a solution containing PCR buffer, MgCl2 and one of four different double-strand DNA-specific binding dyes: SYTO9 [11], SYBR Green I [1], LC Green [10] and SYTO59 (Monis and Rasmussen unpublished). Whilst each of the dyes was used at an optimum concentration, analysing the melting curves under otherwise standard conditions limited any variability that may be contributed by differing reaction conditions or PCR amplification. The comparison of melting curves for each of the Naegleria test amplicons using the four dyes (Figure 3A–D) with melting curve simulations generated by POLAND and MELTSIM showed good agreement in the number of peaks and the peak separation for each of the dyes. The shape of the peaks and peak height were somewhat variable when different dyes were compared, qualitatively most different in the SYBR Green I melting curve (Figure 3B).


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.

Rasmussen JP, Saint CP, Monis PT - BMC Bioinformatics (2007)

Comparison of melting profiles obtained using different intercalating dyes and purified Naegleria amplicons. First derivative plots for purified amplified DNA from Naegleria fowleri (red line), Naegleria lovaniensis (blue line) and Naegleria australiensis (black line) melted in the presence of either SYTO9 (A), SYBR Green (B), LC Green I (C) or SYTO59 (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851973&req=5

Figure 3: Comparison of melting profiles obtained using different intercalating dyes and purified Naegleria amplicons. First derivative plots for purified amplified DNA from Naegleria fowleri (red line), Naegleria lovaniensis (blue line) and Naegleria australiensis (black line) melted in the presence of either SYTO9 (A), SYBR Green (B), LC Green I (C) or SYTO59 (D).
Mentions: To establish that the observed similarity in predicted and actual meting curves was not a "dye-specific" phenomenon, N. australiensis, N. fowleri and N. lovaniensis amplicons were purified and added in equal amounts (ng) to a solution containing PCR buffer, MgCl2 and one of four different double-strand DNA-specific binding dyes: SYTO9 [11], SYBR Green I [1], LC Green [10] and SYTO59 (Monis and Rasmussen unpublished). Whilst each of the dyes was used at an optimum concentration, analysing the melting curves under otherwise standard conditions limited any variability that may be contributed by differing reaction conditions or PCR amplification. The comparison of melting curves for each of the Naegleria test amplicons using the four dyes (Figure 3A–D) with melting curve simulations generated by POLAND and MELTSIM showed good agreement in the number of peaks and the peak separation for each of the dyes. The shape of the peaks and peak height were somewhat variable when different dyes were compared, qualitatively most different in the SYBR Green I melting curve (Figure 3B).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cooperative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Salisbury, SA, Australia. paul.rasmussen@sawater.com.au

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus