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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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Plots of intensity (normalized by signal mean) versus the measured free energy, ΔG°(25°C), for each type of duplex set. (a) Type 1 duplexes containing perfect match w/c base pairs. Correlation coefficients: R = 0.88 (primary set), R = 0.26 (full set); (b) Type 2 duplexes containing mismatches in the center. Correlation coefficients: R = 0.92 (primary set), R = 0.71 (full set); (c) Type 3 duplexes containing multiple mismatches. Correlation coefficients: R = 0.93 (primary set), R = 0.78 (full set). The best linear fit to the primary data set is shown as a dashed line in each case.
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Figure 2: Plots of intensity (normalized by signal mean) versus the measured free energy, ΔG°(25°C), for each type of duplex set. (a) Type 1 duplexes containing perfect match w/c base pairs. Correlation coefficients: R = 0.88 (primary set), R = 0.26 (full set); (b) Type 2 duplexes containing mismatches in the center. Correlation coefficients: R = 0.92 (primary set), R = 0.71 (full set); (c) Type 3 duplexes containing multiple mismatches. Correlation coefficients: R = 0.93 (primary set), R = 0.78 (full set). The best linear fit to the primary data set is shown as a dashed line in each case.

Mentions: Sequences of DNA duplexes examined in experiments are grouped into Type 1, 2 or 3 according to the number and type of mismatches that form in each duplex. Type 1 duplexes contain only perfect-match duplexes (with the exception of duplex 5.1). Duplexes in Type 2 each contain from 2 to 5 mismatches near the middle, while duplexes in Type 3 duplexes contain multiple mismatches interspersed throughout the sequence or mismatches on or near the ends. Correlations between free energy and microarray hybridization intensities were determined for each duplex set. As shown in Figure 2, the relation between ΔG° and intensity is progressively more linear as duplex stability decreases. For the primary data set, duplexes with the most mismatches (Type 3) exhibited the highest degree of linearity (correlation coefficient R = 0.93, = 1.58), while duplexes with no mismatches (Type 1) exhibited the lowest degree of linearity (correlation coefficient R = 0.73, ).Figure 2.


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)

Plots of intensity (normalized by signal mean) versus the measured free energy, ΔG°(25°C), for each type of duplex set. (a) Type 1 duplexes containing perfect match w/c base pairs. Correlation coefficients: R = 0.88 (primary set), R = 0.26 (full set); (b) Type 2 duplexes containing mismatches in the center. Correlation coefficients: R = 0.92 (primary set), R = 0.71 (full set); (c) Type 3 duplexes containing multiple mismatches. Correlation coefficients: R = 0.93 (primary set), R = 0.78 (full set). The best linear fit to the primary data set is shown as a dashed line in each case.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Plots of intensity (normalized by signal mean) versus the measured free energy, ΔG°(25°C), for each type of duplex set. (a) Type 1 duplexes containing perfect match w/c base pairs. Correlation coefficients: R = 0.88 (primary set), R = 0.26 (full set); (b) Type 2 duplexes containing mismatches in the center. Correlation coefficients: R = 0.92 (primary set), R = 0.71 (full set); (c) Type 3 duplexes containing multiple mismatches. Correlation coefficients: R = 0.93 (primary set), R = 0.78 (full set). The best linear fit to the primary data set is shown as a dashed line in each case.
Mentions: Sequences of DNA duplexes examined in experiments are grouped into Type 1, 2 or 3 according to the number and type of mismatches that form in each duplex. Type 1 duplexes contain only perfect-match duplexes (with the exception of duplex 5.1). Duplexes in Type 2 each contain from 2 to 5 mismatches near the middle, while duplexes in Type 3 duplexes contain multiple mismatches interspersed throughout the sequence or mismatches on or near the ends. Correlations between free energy and microarray hybridization intensities were determined for each duplex set. As shown in Figure 2, the relation between ΔG° and intensity is progressively more linear as duplex stability decreases. For the primary data set, duplexes with the most mismatches (Type 3) exhibited the highest degree of linearity (correlation coefficient R = 0.93, = 1.58), while duplexes with no mismatches (Type 1) exhibited the lowest degree of linearity (correlation coefficient R = 0.73, ).Figure 2.

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