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DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison.

Fish DJ, Horne MT, Brewood GP, Goodarzi JP, Alemayehu S, Bhandiwad A, Searles RP, Benight AS - Nucleic Acids Res. (2007)

Bottom Line: Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution.Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes.These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.

View Article: PubMed Central - PubMed

Affiliation: Portland Bioscience, Inc., Portland State University, USA. djf@pdxbio.com

ABSTRACT
Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters DeltaH degrees , DeltaS degrees and DeltaG degrees of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson-Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.

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Comparison of the difference, ΔΔG = ΔG°(mm) − ΔG°(pm), in free energies between perfect match and mismatch duplexes predicted using the nearest-neighbor model and experimentally measured by DSC. (a) Differences in ΔG° between Type 2 and Type 1 duplexes. (b) Differences in ΔG° between Type 3 and Type 1 duplexes. Dark bars (NN) depict ΔΔG predicted with the nearest-neighbor model, assuming ΔG = 0 for tandem mismatches (see text). Light bars (DSC) show ΔΔG as measured by DSC.
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Figure 5: Comparison of the difference, ΔΔG = ΔG°(mm) − ΔG°(pm), in free energies between perfect match and mismatch duplexes predicted using the nearest-neighbor model and experimentally measured by DSC. (a) Differences in ΔG° between Type 2 and Type 1 duplexes. (b) Differences in ΔG° between Type 3 and Type 1 duplexes. Dark bars (NN) depict ΔΔG predicted with the nearest-neighbor model, assuming ΔG = 0 for tandem mismatches (see text). Light bars (DSC) show ΔΔG as measured by DSC.

Mentions: Using analogous expressions for the specific sequences involved, and the published nearest-neighbor stacking stability parameters and tabulated ΔG°loop(n) values, the differences, ΔΔG, between the perfect match duplexes (Type 1) and their corresponding mismatch duplexes (Types 2 and 3) were predicted. The predicted differences were then compared directly with the same differences obtained from experimentally measured free energies. Results are summarized in Figure 5, where predicted differences are plotted as histograms (dark bars) along with DSC-measured quantities (light bars). Note that these are plots of ΔΔG for each mismatch compared to the corresponding perfect match duplex. Thus, the greater the ΔΔG value, the lower the predicted (or measured) stability of the mismatch compared to the perfect match duplex. Nearest-neighbor calculations give somewhat different values when compared to the experimentally measured thermodynamic parameters. This probably results from neglecting enthalpic stabilizing contributions in mismatched regions of the duplex. A taller dark (predicted) bar than light (experimental) bar indicates the mismatch duplex is predicted to be more unstable compared to the perfect match, than experimentally observed. The differences between nearest-neighbor predictions and DSC measurements ranged from −0.94 to 10.28 kcal/mol with an average difference of 5.05 kcal/mol for ΔΔG(Type 2–Type 1), and from 1.94 to 19.25 kcal/mol with an average difference of 10.19 kcal/mol for ΔΔG(Type 3–Type 1).Figure 5.


DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison.

Fish DJ, Horne MT, Brewood GP, Goodarzi JP, Alemayehu S, Bhandiwad A, Searles RP, Benight AS - Nucleic Acids Res. (2007)

Comparison of the difference, ΔΔG = ΔG°(mm) − ΔG°(pm), in free energies between perfect match and mismatch duplexes predicted using the nearest-neighbor model and experimentally measured by DSC. (a) Differences in ΔG° between Type 2 and Type 1 duplexes. (b) Differences in ΔG° between Type 3 and Type 1 duplexes. Dark bars (NN) depict ΔΔG predicted with the nearest-neighbor model, assuming ΔG = 0 for tandem mismatches (see text). Light bars (DSC) show ΔΔG as measured by DSC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175334&req=5

Figure 5: Comparison of the difference, ΔΔG = ΔG°(mm) − ΔG°(pm), in free energies between perfect match and mismatch duplexes predicted using the nearest-neighbor model and experimentally measured by DSC. (a) Differences in ΔG° between Type 2 and Type 1 duplexes. (b) Differences in ΔG° between Type 3 and Type 1 duplexes. Dark bars (NN) depict ΔΔG predicted with the nearest-neighbor model, assuming ΔG = 0 for tandem mismatches (see text). Light bars (DSC) show ΔΔG as measured by DSC.
Mentions: Using analogous expressions for the specific sequences involved, and the published nearest-neighbor stacking stability parameters and tabulated ΔG°loop(n) values, the differences, ΔΔG, between the perfect match duplexes (Type 1) and their corresponding mismatch duplexes (Types 2 and 3) were predicted. The predicted differences were then compared directly with the same differences obtained from experimentally measured free energies. Results are summarized in Figure 5, where predicted differences are plotted as histograms (dark bars) along with DSC-measured quantities (light bars). Note that these are plots of ΔΔG for each mismatch compared to the corresponding perfect match duplex. Thus, the greater the ΔΔG value, the lower the predicted (or measured) stability of the mismatch compared to the perfect match duplex. Nearest-neighbor calculations give somewhat different values when compared to the experimentally measured thermodynamic parameters. This probably results from neglecting enthalpic stabilizing contributions in mismatched regions of the duplex. A taller dark (predicted) bar than light (experimental) bar indicates the mismatch duplex is predicted to be more unstable compared to the perfect match, than experimentally observed. The differences between nearest-neighbor predictions and DSC measurements ranged from −0.94 to 10.28 kcal/mol with an average difference of 5.05 kcal/mol for ΔΔG(Type 2–Type 1), and from 1.94 to 19.25 kcal/mol with an average difference of 10.19 kcal/mol for ΔΔG(Type 3–Type 1).Figure 5.

Bottom Line: Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution.Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes.These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.

View Article: PubMed Central - PubMed

Affiliation: Portland Bioscience, Inc., Portland State University, USA. djf@pdxbio.com

ABSTRACT
Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters DeltaH degrees , DeltaS degrees and DeltaG degrees of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson-Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.

Show MeSH