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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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Charts showing relative effect of mismatches on stability and microarray intensity. Results are grouped according to probe number, and each type of duplex: Type 1 (perfect match), Types 2 and 3 are represented by a different histograms. Solution results are shown in the upper subplot, and microarray results are shown in the lower subplot.
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Figure 4: Charts showing relative effect of mismatches on stability and microarray intensity. Results are grouped according to probe number, and each type of duplex: Type 1 (perfect match), Types 2 and 3 are represented by a different histograms. Solution results are shown in the upper subplot, and microarray results are shown in the lower subplot.

Mentions: In order to analyze effects of mismatched bases on ΔG° in solution, and their concomitant effects on microarray hybridization intensities, experimental values obtained for each type of duplex were directly compared. In Figure 4, solution and microarray results are displayed in separate subplots. In each subplot, results are grouped according to probe number (1–10), and each type of duplex (Type 1—perfect match, Type 2 and Type 3) is represented as a histogram. Several interesting points arise from this analysis.Figure 4.


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)

Charts showing relative effect of mismatches on stability and microarray intensity. Results are grouped according to probe number, and each type of duplex: Type 1 (perfect match), Types 2 and 3 are represented by a different histograms. Solution results are shown in the upper subplot, and microarray results are shown in the lower subplot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Charts showing relative effect of mismatches on stability and microarray intensity. Results are grouped according to probe number, and each type of duplex: Type 1 (perfect match), Types 2 and 3 are represented by a different histograms. Solution results are shown in the upper subplot, and microarray results are shown in the lower subplot.
Mentions: In order to analyze effects of mismatched bases on ΔG° in solution, and their concomitant effects on microarray hybridization intensities, experimental values obtained for each type of duplex were directly compared. In Figure 4, solution and microarray results are displayed in separate subplots. In each subplot, results are grouped according to probe number (1–10), and each type of duplex (Type 1—perfect match, Type 2 and Type 3) is represented as a histogram. Several interesting points arise from this analysis.Figure 4.

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