Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparison of change in enthalpy (ΔΔH), relative to the perfect match and change in entropy (TΔΔS, T = 25°C), relative to the perfect match, as measured by DSC, for both Type 2 (squares) and Type 3 (triangles) duplexes. The dashed line that fits the data has a slope m = 0.83 and intercept b = −1.22, with a linear correlation coefficient, R = 0.997. For reference, the dotted line y = x is also shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175334&req=5

Figure 6: Comparison of change in enthalpy (ΔΔH), relative to the perfect match and change in entropy (TΔΔS, T = 25°C), relative to the perfect match, as measured by DSC, for both Type 2 (squares) and Type 3 (triangles) duplexes. The dashed line that fits the data has a slope m = 0.83 and intercept b = −1.22, with a linear correlation coefficient, R = 0.997. For reference, the dotted line y = x is also shown.

Mentions: For the sets of duplexes examined, the only difference between Type 1 and Type 2 or Type 1 and Type 3, for a given probe number (1–10), is the introduction of tandem mismatches in various positions. Therefore, thermodynamic effects of substituting w/c base pairs with tandem mismatches in the same duplex sequence can be directly assessed. Relative differences of measured ΔH° and ΔS° values for each probe number, and for each mismatch type (Types 2 and 3) are plotted in Figure 6. As shown, a direct correlation was found (correlation coefficient R = 0.997) between the changes in enthalpy, ΔΔH = ΔH°(mm) − ΔH°(pm), and entropy, TΔΔS = T[ΔS°(mm) − ΔS°(pm)], relative to the perfect match, regardless of the number, type or mismatch position (at T = 25°C). The best linear fit y = mx + b to the data has parameters m = 0.83 and b = −1.22, and is shown as a dashed line in Figure 6. The line y = x is shown as a dotted line for reference. When comparing mismatched duplexes to the corresponding perfectly matched duplexes, it is clear that the loss of stabilizing interactions is not fully balanced by a gain in degrees of freedom of the system. This comparison for tandem mismatches expands previous results in the literature reporting ‘free energy compensation’ (cf. 19) for perfect match duplexes or duplexes containing only single base pair mismatches.Figure 6.


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 change in enthalpy (ΔΔH), relative to the perfect match and change in entropy (TΔΔS, T = 25°C), relative to the perfect match, as measured by DSC, for both Type 2 (squares) and Type 3 (triangles) duplexes. The dashed line that fits the data has a slope m = 0.83 and intercept b = −1.22, with a linear correlation coefficient, R = 0.997. For reference, the dotted line y = x is also shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Comparison of change in enthalpy (ΔΔH), relative to the perfect match and change in entropy (TΔΔS, T = 25°C), relative to the perfect match, as measured by DSC, for both Type 2 (squares) and Type 3 (triangles) duplexes. The dashed line that fits the data has a slope m = 0.83 and intercept b = −1.22, with a linear correlation coefficient, R = 0.997. For reference, the dotted line y = x is also shown.
Mentions: For the sets of duplexes examined, the only difference between Type 1 and Type 2 or Type 1 and Type 3, for a given probe number (1–10), is the introduction of tandem mismatches in various positions. Therefore, thermodynamic effects of substituting w/c base pairs with tandem mismatches in the same duplex sequence can be directly assessed. Relative differences of measured ΔH° and ΔS° values for each probe number, and for each mismatch type (Types 2 and 3) are plotted in Figure 6. As shown, a direct correlation was found (correlation coefficient R = 0.997) between the changes in enthalpy, ΔΔH = ΔH°(mm) − ΔH°(pm), and entropy, TΔΔS = T[ΔS°(mm) − ΔS°(pm)], relative to the perfect match, regardless of the number, type or mismatch position (at T = 25°C). The best linear fit y = mx + b to the data has parameters m = 0.83 and b = −1.22, and is shown as a dashed line in Figure 6. The line y = x is shown as a dotted line for reference. When comparing mismatched duplexes to the corresponding perfectly matched duplexes, it is clear that the loss of stabilizing interactions is not fully balanced by a gain in degrees of freedom of the system. This comparison for tandem mismatches expands previous results in the literature reporting ‘free energy compensation’ (cf. 19) for perfect match duplexes or duplexes containing only single base pair mismatches.Figure 6.

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
Related in: MedlinePlus