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
Comparisons of microarray hybridization and solution melting. (a) Plot of intensities (normalized by signal mean) of the full data set collected from 63 experiments versus the primary microarray data set comprised of results from 11 experiments. The dashed line shows the best linear fit to the data (R = 0.75). (b) Plot of the primary data set of relative microarray intensities versus the measured free energy, ΔG°(25°C). The dashed line is the best linear fit to the data (R = 0.92). (c) Plot of the full data set of the relative microarray intensity versus the measured free energy, ΔG°(25°C). The dotted line is the same line as in (b) obtained for the best fit to the primary data set. The black dashed line is the best linear fit to the full data set (R = 0.53). Upper and lower dashed lines depict the parameter window bounded by the line y = m·ΔG° + b, with m = 0.250 ± 0.007 and b = 4.86 ± 1.50.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Comparisons of microarray hybridization and solution melting. (a) Plot of intensities (normalized by signal mean) of the full data set collected from 63 experiments versus the primary microarray data set comprised of results from 11 experiments. The dashed line shows the best linear fit to the data (R = 0.75). (b) Plot of the primary data set of relative microarray intensities versus the measured free energy, ΔG°(25°C). The dashed line is the best linear fit to the data (R = 0.92). (c) Plot of the full data set of the relative microarray intensity versus the measured free energy, ΔG°(25°C). The dotted line is the same line as in (b) obtained for the best fit to the primary data set. The black dashed line is the best linear fit to the full data set (R = 0.53). Upper and lower dashed lines depict the parameter window bounded by the line y = m·ΔG° + b, with m = 0.250 ± 0.007 and b = 4.86 ± 1.50.

Mentions: In analyzing microarray intensity data, three sources of signal variation are typically encountered: chip-to-chip signal variation, signal variation within each chip and signal variation between different print runs. Being aware of the potential sources for signal variation, initial data analysis was performed on results obtained from a subset of 11 microarray experiments that were shown to have relatively small variations of each type. These 11 experiments were identified by constructing a correlation coefficient matrix for the full data set to compare replicate probe signals for all the data (data not shown). The sets of data with the highest correlation coefficients, larger than 0.8, comprised the ‘primary data set’. The correlation coefficient matrix also showed that two print runs were statistically robust and gave consistent replicate data. Given the current experimental variability of the spotted microarray process, this analysis demonstrates the importance of careful statistical analysis of spot-to-spot reproducibility and chip-to-chip variability for microarray data when quantitative results are desired. The complete set of results from all 63 experiments is referred to as the ‘full data set’. A direct comparison of microarray results from the primary and full data sets is shown in Figure 1a. Thermodynamic parameters measured in solution by DSC are displayed in Table 2. Results from microarray experiments for both the primary and full data sets are shown in Table 3.Figure 1.


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)

Comparisons of microarray hybridization and solution melting. (a) Plot of intensities (normalized by signal mean) of the full data set collected from 63 experiments versus the primary microarray data set comprised of results from 11 experiments. The dashed line shows the best linear fit to the data (R = 0.75). (b) Plot of the primary data set of relative microarray intensities versus the measured free energy, ΔG°(25°C). The dashed line is the best linear fit to the data (R = 0.92). (c) Plot of the full data set of the relative microarray intensity versus the measured free energy, ΔG°(25°C). The dotted line is the same line as in (b) obtained for the best fit to the primary data set. The black dashed line is the best linear fit to the full data set (R = 0.53). Upper and lower dashed lines depict the parameter window bounded by the line y = m·ΔG° + b, with m = 0.250 ± 0.007 and b = 4.86 ± 1.50.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Comparisons of microarray hybridization and solution melting. (a) Plot of intensities (normalized by signal mean) of the full data set collected from 63 experiments versus the primary microarray data set comprised of results from 11 experiments. The dashed line shows the best linear fit to the data (R = 0.75). (b) Plot of the primary data set of relative microarray intensities versus the measured free energy, ΔG°(25°C). The dashed line is the best linear fit to the data (R = 0.92). (c) Plot of the full data set of the relative microarray intensity versus the measured free energy, ΔG°(25°C). The dotted line is the same line as in (b) obtained for the best fit to the primary data set. The black dashed line is the best linear fit to the full data set (R = 0.53). Upper and lower dashed lines depict the parameter window bounded by the line y = m·ΔG° + b, with m = 0.250 ± 0.007 and b = 4.86 ± 1.50.
Mentions: In analyzing microarray intensity data, three sources of signal variation are typically encountered: chip-to-chip signal variation, signal variation within each chip and signal variation between different print runs. Being aware of the potential sources for signal variation, initial data analysis was performed on results obtained from a subset of 11 microarray experiments that were shown to have relatively small variations of each type. These 11 experiments were identified by constructing a correlation coefficient matrix for the full data set to compare replicate probe signals for all the data (data not shown). The sets of data with the highest correlation coefficients, larger than 0.8, comprised the ‘primary data set’. The correlation coefficient matrix also showed that two print runs were statistically robust and gave consistent replicate data. Given the current experimental variability of the spotted microarray process, this analysis demonstrates the importance of careful statistical analysis of spot-to-spot reproducibility and chip-to-chip variability for microarray data when quantitative results are desired. The complete set of results from all 63 experiments is referred to as the ‘full data set’. A direct comparison of microarray results from the primary and full data sets is shown in Figure 1a. Thermodynamic parameters measured in solution by DSC are displayed in Table 2. Results from microarray experiments for both the primary and full data sets are shown in Table 3.Figure 1.

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