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Analysis of multidimensional G-quadruplex melting curves.

ABSTRACT: Multidimensional "3D" melting curves for G-quadruplexes are obtained by recording whole spectra (absorbance, CD, fluorescence) as a function of temperature, rather than the common approach of recording the spectral response to temperature at a single wavelength. 3D melting curves are richer in information, and can be used to enumerate the number of significant species and intermediate states required to properly analyze the thermal denaturation reaction to obtain thermodynamic information. This unit describes the application of the method of singular value decomposition to the analysis of 3D melting data obtained for G-quadruplex structures, and how the results of such an analysis can be used to provide a more complete characterization of the mechanism of quadruplex unfolding.

SUBMITTER: Gray RD

PROVIDER: S-EPMC3398426 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Analysis of multidimensional G-quadruplex melting curves.

Gray Robert D RD Chaires Jonathan B JB

Current protocols in nucleic acid chemistry 20110601

Multidimensional "3D" melting curves for G-quadruplexes are obtained by recording whole spectra (absorbance, CD, fluorescence) as a function of temperature, rather than the common approach of recording the spectral response to temperature at a single wavelength. 3D melting curves are richer in information, and can be used to enumerate the number of significant species and intermediate states required to properly analyze the thermal denaturation reaction to obtain thermodynamic information. This ...[more]

PMID: 21638271

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Project description: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.

Dataset Information

Analysis of multidimensional G-quadruplex melting curves.

Publications

Analysis of multidimensional G-quadruplex melting curves.

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