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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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Change in enthalpy, ΔΔH (dark), and change in entropy, TΔΔS, T = 25°C (light), for five specific duplexes. Type 2 duplexes (6.2, 7.2 and 10.2) have a GA/AG mismatch in the middle; Type 3 duplexes (1.3 and 3.3) have a GA/AG mismatch near the end.
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Figure 8: Change in enthalpy, ΔΔH (dark), and change in entropy, TΔΔS, T = 25°C (light), for five specific duplexes. Type 2 duplexes (6.2, 7.2 and 10.2) have a GA/AG mismatch in the middle; Type 3 duplexes (1.3 and 3.3) have a GA/AG mismatch near the end.

Mentions: Duplexes 6.2, 7.2 and 10.2 all contain a GA/AG mismatch near the middle (Table 1, column 2), while duplexes 1.3 (and 3.3) each contain a GA/AG (AG/GA) mismatch at (or near) the end (Table 1, column 3). For these specific sequences, differences in ΔΔH and TΔΔS indicate effects of relative position and nearest-neighbor flanking sequences on thermodynamics of duplex formation. The change in entropy and enthalpy for these duplexes (relative to the perfect match) is shown in Figure 8. Note that the increasing trend among Type 2 duplexes (7.2 < 6.2 < 10.2) cannot be due to GC content alone, since duplexes 6.2 and 7.2 have similar percentage of GC (23.1 and 26.9%, respectively). However, examination of the base pairs flanking the tandem mismatch in each case reveals that the flanking base pairs in 7.2 are both G/C, in 6.2 one is a G/C and the other an A/T and in 10.2 both are A/T base pairs. Thus, the relative stability of flanking base pairs accounts for the change in entropy and enthalpy for these three duplexes.Figure 8.


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)

Change in enthalpy, ΔΔH (dark), and change in entropy, TΔΔS, T = 25°C (light), for five specific duplexes. Type 2 duplexes (6.2, 7.2 and 10.2) have a GA/AG mismatch in the middle; Type 3 duplexes (1.3 and 3.3) have a GA/AG mismatch near the end.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Change in enthalpy, ΔΔH (dark), and change in entropy, TΔΔS, T = 25°C (light), for five specific duplexes. Type 2 duplexes (6.2, 7.2 and 10.2) have a GA/AG mismatch in the middle; Type 3 duplexes (1.3 and 3.3) have a GA/AG mismatch near the end.
Mentions: Duplexes 6.2, 7.2 and 10.2 all contain a GA/AG mismatch near the middle (Table 1, column 2), while duplexes 1.3 (and 3.3) each contain a GA/AG (AG/GA) mismatch at (or near) the end (Table 1, column 3). For these specific sequences, differences in ΔΔH and TΔΔS indicate effects of relative position and nearest-neighbor flanking sequences on thermodynamics of duplex formation. The change in entropy and enthalpy for these duplexes (relative to the perfect match) is shown in Figure 8. Note that the increasing trend among Type 2 duplexes (7.2 < 6.2 < 10.2) cannot be due to GC content alone, since duplexes 6.2 and 7.2 have similar percentage of GC (23.1 and 26.9%, respectively). However, examination of the base pairs flanking the tandem mismatch in each case reveals that the flanking base pairs in 7.2 are both G/C, in 6.2 one is a G/C and the other an A/T and in 10.2 both are A/T base pairs. Thus, the relative stability of flanking base pairs accounts for the change in entropy and enthalpy for these three duplexes.Figure 8.

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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