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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 Cryptosporidium species. Plots (solid lines) of the first derivatives (for either fluorescence, theta or absorbance) versus temperature for Cryptosporidium parvum (A, B, C) and Cryptosporidium muris (D, E, F) for either DNA amplified and melted in the presence of SYTO9 (A, D) or melting simulations conducted using the amplicon DNA sequences and either MELTSIM (B, E) or POLAND (C, F) 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 2: Comparison of real-time PCR and simulated melting profiles for different Cryptosporidium species. Plots (solid lines) of the first derivatives (for either fluorescence, theta or absorbance) versus temperature for Cryptosporidium parvum (A, B, C) and Cryptosporidium muris (D, E, F) for either DNA amplified and melted in the presence of SYTO9 (A, D) or melting simulations conducted using the amplicon DNA sequences and either MELTSIM (B, E) or POLAND (C, F) 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: A segment of 18S rDNA from Cryptosporidium parvum and Cryptosporidium muris, approximately 300 bp, was amplified and melted in the presence of SYTO9. The differentiated melting curves resulted in complex profiles that allowed the ready differentiation of these two species, with C. muris producing 2 peaks with Tms of 75.5°C and 83.5°C (Figure 2D) and C. parvum producing a peak at 73.8°C and a peak with a shoulder at 80.8°C and 83.0°C respectively (Figure 2A). The standard deviation in Tm observed between replicates (n = 4 for each species) was less than 0.3°C (data not shown). In the case of the C. parvum sequence there was a subtle difference between the POLAND (Figure 2C) and MELTSIM (Figure 2B) predictions, with a difference between the position and height of the second peak and its shoulder. The MELTSIM profile more closely matched the observed profile (Figure 2A). The melting profiles predicted C. muris by MELTSIM (Figure 2E) and POLAND (Figure 2F) were the same as each other and similar to the observed profile (Figure 2D) in terms of the number of peaks and spacing between peaks. In both cases the peak heights and absolute values Tms differed between the in vitro and in silico profiles. The melt maps predicted by each program were similar to each other for C. parvum (Figure 2B, 2C) and C. muris (Figure 2E, F); however, the MELTSIM maps were again truncated, here at 238 bp.


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 Cryptosporidium species. Plots (solid lines) of the first derivatives (for either fluorescence, theta or absorbance) versus temperature for Cryptosporidium parvum (A, B, C) and Cryptosporidium muris (D, E, F) for either DNA amplified and melted in the presence of SYTO9 (A, D) or melting simulations conducted using the amplicon DNA sequences and either MELTSIM (B, E) or POLAND (C, F) 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 2: Comparison of real-time PCR and simulated melting profiles for different Cryptosporidium species. Plots (solid lines) of the first derivatives (for either fluorescence, theta or absorbance) versus temperature for Cryptosporidium parvum (A, B, C) and Cryptosporidium muris (D, E, F) for either DNA amplified and melted in the presence of SYTO9 (A, D) or melting simulations conducted using the amplicon DNA sequences and either MELTSIM (B, E) or POLAND (C, F) 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: A segment of 18S rDNA from Cryptosporidium parvum and Cryptosporidium muris, approximately 300 bp, was amplified and melted in the presence of SYTO9. The differentiated melting curves resulted in complex profiles that allowed the ready differentiation of these two species, with C. muris producing 2 peaks with Tms of 75.5°C and 83.5°C (Figure 2D) and C. parvum producing a peak at 73.8°C and a peak with a shoulder at 80.8°C and 83.0°C respectively (Figure 2A). The standard deviation in Tm observed between replicates (n = 4 for each species) was less than 0.3°C (data not shown). In the case of the C. parvum sequence there was a subtle difference between the POLAND (Figure 2C) and MELTSIM (Figure 2B) predictions, with a difference between the position and height of the second peak and its shoulder. The MELTSIM profile more closely matched the observed profile (Figure 2A). The melting profiles predicted C. muris by MELTSIM (Figure 2E) and POLAND (Figure 2F) were the same as each other and similar to the observed profile (Figure 2D) in terms of the number of peaks and spacing between peaks. In both cases the peak heights and absolute values Tms differed between the in vitro and in silico profiles. The melt maps predicted by each program were similar to each other for C. parvum (Figure 2B, 2C) and C. muris (Figure 2E, F); however, the MELTSIM maps were again truncated, here at 238 bp.

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