dnaMATE server


	This is a server that calculates the annealing or melting temperature of any given short DNA sequence (in the range of 16-30 nts) using five different approximations. A merged or consensus temperature among all calculations is also given. In addition to this, the server will inform to the user about the expected variation of the melting temperature estimation, which depends on the specific oligonucleotide sequence. In a recent study (Panjkovich and Melo, 2005), we have demonstrated that large variations in the melting temperature estimations are observed among different methods. The magnitude of the observed differences depends on the length and CG-content of the oligonucleotide sequence, but it cannot be explained in a trivial manner. In fact, complex relationships are observed: in some cases two or more methods give similar melting temperature values (within 5° C) for a significant fraction (greater than 80%) of a large set of oligonucleotides (2000 different sequences) of fixed length and similar CG-content, but it turns out that these values are finally highly uncorrelated. Based on these results, we suggest that additional experimental data is required in order to eliminate the existing bias of the current methods and parameters towards oligonucleotide length and composition (most of the methods have been parameterized based on a very restricted set of oligos for which experimental data is currently available). Meanwhile, the best solution to avoid a large error in the melting temperature estimation of a given oligonucleotide sequence is to attempt to get a consensus value among the existing methods that consistently show similar values for a given length and CG content. This is what this server attempts to do, based on the large scale comparative study that we have performed. Accurate melting temperature estimation could not be highly relevant for a classic single-locus PCR, but it becomes extremely important in other applications such as multiplex PCR, quantitative PCR and the design of fixed short length oligonucleotide microarrays (Affymetrix-like chips), where dozens or thousands of DNA molecules are used simultaneously under identical experimental conditions and, of course, at a single and fixed temperature. The calculations performed in the consensus Tm estimation method are described in the Figure below. For details about the thermodynamic calculations see the Methods section. of supplemental material. (Top panel) The consensus map from the previous comparative benchmark (Panjkovich and Melo, 2005) is illustrated. In this benchmark, three thermodynamic data sets were compared: Bre stands for Breslauer et al. 1986; San stands for SantaLucia et al. 1996; Sug stands for Sugimoto et al. 1996. In this map, four distinct regions were obtained: 1) simultaneously, Bre and Sug on the one hand, and San and Sug on the other, exhibited similar Tm values (white colour); 2) only Bre and Sug exhibited similar Tm values (light gray colour); 3) only San and Sug exhibited similar Tm values (dark gray colour); and finally, 4) no consensus was observed among any of the methods (black colour). Bre and San did not show a similar behavior in the complete range of sequence length and percentage of CG-content. (Bottom panel) A graphical illustration of the different consensus map zones is shown. Each method is represented as a particular side of an equilateral triangle and the intersection among methods is shown with the corresponding color of the consensus map. The mathematical expressions used to calculate the consensus Tm at each zone are also indicated. In the case of San calculations the most recent thermodynamic parameters (SantaLucia, 1998) are being used by the server to calculate the consensus melting temperature. This modification respect to our previous study (Melo and Panjkovich, 2005) has improved even more the accuracy of this server. The Tm estimations of oligonucleotides falling into the black regions of the consensus map by any of the methods could have a large error. The Tm estimation error at the other regions where some consensus was observed is expected to be small (below 3-5 ºC).

	There are three different methods that can be used to calculate the melting temperature of short DNA sequences. The first one is called the 'Basic' method (bas), which estimates the melting temperature only based on the composition of nucleotides. The second method is called 'Salt Adjusted' (sal), which is similar to the previous method but includes a logarithmic factor to correct for salt concentration. The third method is called the 'Thermodynamic' method, which uses the nearest-neighbor model and the experimental values of enthalpy and entropy to get the free energy of duplex formation. This method also includes additional terms to correct the melting temperature estimation for the effects of oligo and salt concentration. Currently, there are three thermodynamic tables that have been published in the literature and are widely used for primer melting temperature calculation using the nearest neighbor approach. These are the Breslauer (Breslauer et al., 1986) table (defined as Th1), the Santalucia (Santalucia, 1998) table (defined as Th2), and the Sugimoto (Sugimoto et al., 1996) table (defined as Th3). This server will calculate the melting temperature of one or more oligonucleotides by using the three different methods and these existing tables of thermodynamic parameters mentioned above, thus giving five different melting temperature values. In addition to this, a consensus or merged annealing temperature based on the three thermodynamic sets will be also given, which is expected to have the smaller error in the long run (average error of several independent estimations). A detailed description of the mathematical expressions and experimental data tables used to calculate the melting temperature of each method is provided here.

	For detailed information about the comparative study we have carried out, read the following paper: Panjkovich and Melo (2005). For additional data, check out the following link containing some supplementary material.

	Panjkovich, A., Norambuena, T. and Melo, F. (2005) dnaMATE: a consensus melting temperature prediction server for short DNA sequences. Nucleic Acids Research, 33, 570-572. Panjkovich, A. and Melo, F. (2005) Comparison of DNA melting temperature calculation methods for short DNA sequences. Bioinformatics* 21, 711-722.* Breslauer, K.J., Frank,R., Blöcker, H. and Marky, L.A. (1986) Predicting DNA Duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746-3750. SantaLucia , Jr. J., Allawi, H.T. and Seneviratne, P.A. (1996) Improved Nearest-Neighbor Parameters for Predicting DNA Duplex Stability.Biochemistry 35, 3555-3562. SantaLucia, Jr. J. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 95, 1460-1465. Sugimoto , N., Nakano, S., Yoneyama, M. and Honda, K. (1996) Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes.Nucleic Acids Res. 24, 4501-4505.