Influence of buffer species on the thermodynamics of short DNA duplex melting: sodium phosphate versus sodium cacodylate.
Thermodynamic parameters of the melting transitions of 53 short duplex DNAs were experimentally evaluated by differential scanning calorimetry melting curve analysis. Solvents for the DNA solutions contained approximately 1 M Na+ and either 10 mM cacodylate or phosphate buffer. Thermodynamic parameters obtained in the two solvent environments were compared and quantitatively assessed. Thermodynamic stabilities (deltaG(o) (25 degrees C)) of the duplexes studied ranged from quite stable perfect match duplexes (approximately -30 kcal/mol) to relatively unstable mismatch duplexes (approximately -9 kcal/mol) and ranged in length from 18 to 22 basepairs. A significant difference in stability (average free energy difference of approximately 3 kcal/mol) was found for all duplexes melted in phosphate (greater stability) versus cacodylate buffers. Measured effects of buffer species appear to be relatively unaffected by duplex length or sequence content. The popular sets of published nearest-neighbor (n-n) stability parameters for Watson-Crick (w/c) and single-base mismatches were evaluated from melting studies performed in cacodylate buffer (SantaLucia and Hicks, Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 415). Thus, when using these parameters to make predictions of sequence dependent stability of DNA oligomers in buffers other than cacodylate (e.g., phosphate) one should be mindful that in addition to sodium ion concentration, the type of buffer species also provides a minor but significant contribution to duplex stability. Such considerations could potentially influence results of sequence dependent analysis using published n-n parameters and impact results of thermodynamic calculations. Such calculations and analyses are typically employed in the design and interpretation of DNA multiplex hybridization experiments.
Alemayehu
Saba
S
Portland Bioscience Inc., 2828 SW Corbett Ave., Suite 116, Portland, Oregon 97201, USA.
Fish
Daniel J
DJ
Brewood
Greg P
GP
Horne
M Todd
MT
Manyanga
Fidelis
F
Dickman
Rebekah
R
Yates
Ian
I
Benight
Albert S
AS
D001483:Base Sequence; D002021:Buffers; D002101:Cacodylic Acid / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning / Q000379:methods; D004247:DNA / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry*; D012964:Sodium / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013816:Thermodynamics
D002021:Buffers; D010710:Phosphates; D012997:Solvents; D004247:DNA; D012964:Sodium; D002101:Cacodylic Acid; C018279:sodium phosphate