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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 real-time PCR and simulated melting profiles for different Naegleria species. Plots (solid lines) of the first derivatives (for either fluorescence, theta or absorbance) versus temperature for Naegleria fowleri (A, B, C), Naegleria lovaniensis (D, E, F) and Naegleria australiensis (G, H, I) for either DNA amplified and melted in the presence of SYTO9 (A, D, G) or melting simulations conducted using the amplicon DNA sequences and either MELTSIM (B, E, H) or POLAND (C, F, I) respectively. The melt map for each sequence (base position versus predicted Tm) determined by MELTSIM is indicated by magenta circles. The equivalent profiles calculated by POLAND are indicated by blue plus signs or red squares for first order and second order reactions respectively.
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Figure 1: Comparison of real-time PCR and simulated melting profiles for different Naegleria species. Plots (solid lines) of the first derivatives (for either fluorescence, theta or absorbance) versus temperature for Naegleria fowleri (A, B, C), Naegleria lovaniensis (D, E, F) and Naegleria australiensis (G, H, I) for either DNA amplified and melted in the presence of SYTO9 (A, D, G) or melting simulations conducted using the amplicon DNA sequences and either MELTSIM (B, E, H) or POLAND (C, F, I) respectively. The melt map for each sequence (base position versus predicted Tm) determined by MELTSIM is indicated by magenta circles. The equivalent profiles calculated by POLAND are indicated by blue plus signs or red squares for first order and second order reactions respectively.

Mentions: The SYTO9 melting curves of amplicons from the intergenic spacer region of N. australiensis, N. fowleri and N. lovaniensis were very distinct as previously reported [12], with multiple peaks differing in shape and height: the N. australiensis amplicon melted with a single sharp peak (Fig 1G); the N. fowleri amplicon melted with three peaks all at different heights (Fig 1A); and the N. lovaniensis amplicon melted with two peaks, the first approximately double the amplitude of the second (Figure 1D). When the sequence of each Naegleria amplicon was subjected to a melting simulation using the POLAND (Figure 1C, F, I) and MELTSIM (Figure 1B, E, H) programs, the predicted melting curves were similar to the profiles obtained using SYTO9. The number of melt peaks and the separation of these peaks were consistent between the simulation programs and the physical data resulting from real-time PCR melting curve analysis. The The predicted relative peak heights (height of a given peak relative to the other peaks for a given template) for N. fowleri and N. lovaniensis did not exactly match the physical data (Figure 1A, D), although the MELTSIM profiles (Figure 1B, E) appeared to be a closer prediction compared to POLAND (Figure 1C, F). The values of the Tms predicted by either program did not match the Tms obtained by DNA melting curve analysis using SYTO9. The melting maps (Tm predicted for each base position (or domain) versus base position) were similar for the POLAND first order reaction (Fig 1C, F) and MELTSIM (Figure 1B, E) predictions for N. fowleri and N. lovaniensis, but slightly different for N. australiensis, particularly in the region at around 250 bp. In the case of MELTSIM, the melting map did not cover the entire amplicon and appeared to be truncated at 357 bp for N. fowleri and 298 bp for N. lovaniensis and N. australiensis.


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 real-time PCR and simulated melting profiles for different Naegleria species. Plots (solid lines) of the first derivatives (for either fluorescence, theta or absorbance) versus temperature for Naegleria fowleri (A, B, C), Naegleria lovaniensis (D, E, F) and Naegleria australiensis (G, H, I) for either DNA amplified and melted in the presence of SYTO9 (A, D, G) or melting simulations conducted using the amplicon DNA sequences and either MELTSIM (B, E, H) or POLAND (C, F, I) respectively. The melt map for each sequence (base position versus predicted Tm) determined by MELTSIM is indicated by magenta circles. The equivalent profiles calculated by POLAND are indicated by blue plus signs or red squares for first order and second order reactions respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Comparison of real-time PCR and simulated melting profiles for different Naegleria species. Plots (solid lines) of the first derivatives (for either fluorescence, theta or absorbance) versus temperature for Naegleria fowleri (A, B, C), Naegleria lovaniensis (D, E, F) and Naegleria australiensis (G, H, I) for either DNA amplified and melted in the presence of SYTO9 (A, D, G) or melting simulations conducted using the amplicon DNA sequences and either MELTSIM (B, E, H) or POLAND (C, F, I) respectively. The melt map for each sequence (base position versus predicted Tm) determined by MELTSIM is indicated by magenta circles. The equivalent profiles calculated by POLAND are indicated by blue plus signs or red squares for first order and second order reactions respectively.
Mentions: The SYTO9 melting curves of amplicons from the intergenic spacer region of N. australiensis, N. fowleri and N. lovaniensis were very distinct as previously reported [12], with multiple peaks differing in shape and height: the N. australiensis amplicon melted with a single sharp peak (Fig 1G); the N. fowleri amplicon melted with three peaks all at different heights (Fig 1A); and the N. lovaniensis amplicon melted with two peaks, the first approximately double the amplitude of the second (Figure 1D). When the sequence of each Naegleria amplicon was subjected to a melting simulation using the POLAND (Figure 1C, F, I) and MELTSIM (Figure 1B, E, H) programs, the predicted melting curves were similar to the profiles obtained using SYTO9. The number of melt peaks and the separation of these peaks were consistent between the simulation programs and the physical data resulting from real-time PCR melting curve analysis. The The predicted relative peak heights (height of a given peak relative to the other peaks for a given template) for N. fowleri and N. lovaniensis did not exactly match the physical data (Figure 1A, D), although the MELTSIM profiles (Figure 1B, E) appeared to be a closer prediction compared to POLAND (Figure 1C, F). The values of the Tms predicted by either program did not match the Tms obtained by DNA melting curve analysis using SYTO9. The melting maps (Tm predicted for each base position (or domain) versus base position) were similar for the POLAND first order reaction (Fig 1C, F) and MELTSIM (Figure 1B, E) predictions for N. fowleri and N. lovaniensis, but slightly different for N. australiensis, particularly in the region at around 250 bp. In the case of MELTSIM, the melting map did not cover the entire amplicon and appeared to be truncated at 357 bp for N. fowleri and 298 bp for N. lovaniensis and N. australiensis.

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