Microarray technology is based on the simple yet efficient priniciple of DNA hybridization. In each spot on a microarray slide, oligonucleotide or cDNA probes complementary to a specific gene sequence are attached. These probes measure the expression level for the corresponding gene by capturing labelled RNA or cDNA from a particular tissue sample. Hybridization and washing conditions are chosen such that the probe hybridizes with only the target sequence molecules with high efficiency and stability, while, all cross hybridizations are washed away. Therefore, estimation of DNA melting temperature for both self hybridization and all possible cross hybridizations is critical in optimal oligo design. Sarani estimates DNA melting temperatures considering DNA Nearest-Neighbour thermodynamics energy parameters using the following equation from SantaLucia (1998; Proc. Natl. Acad. Sci. USA 95, 1460-1465).

Tm = {(DH * 1000) / (DS + R * ln(C T )) } - 273.14
where,
DH = Total DH (Enthalpy of duplex formation) in kcal / mol
DS = DS(Oligomer, [NA + ]) (with appropriate salt correction)
R = gas constant ( 1.987 cal/K.mol)
C T = oligonucleotide concentration (in mol)

Salt Correction: Cations like Na + , Mg ++ present in the solution stabilize the DNA duplex and affect the entropy and free energy of the system. Therefore, it is essential to make a salt dependent correction to DG and DS. It has been assumed that salt correction for the DNA is independent of nucleotide sequence but dependent on the oligonucleotide length (SantaLucia, 1998).

Salt Correction: Cations like Na + , Mg ++ present in the solution stabilize the DNA duplex and affect the entropy and free energy of the system. Therefore, it is essential to make a salt dependent correction to DG and DS. It has been assumed that salt correction for the DNA is independent of nucleotide sequence but dependent on the oligonucleotide length (SantaLucia, 1998).

DS (Oligomer, [Na + ]) = DS(Unified Oligomer, 1M NaCl) + 0.368 * N * ln[Na+] where, N = (length of oligonucleotide - 1) ln[Na + ] = natural log of Sodium ion concentration

Note: In a typical microarray experiment, the concentration of oligonucleotides spotted on the chip or the concentration of the complementary target cDNA molecules in the sample is not known. In fact the objective of the microarray experiments is to measure the concentration of target cDNA molecules in a sample. Sarani allows rough concentrations to be specified (default conc., 10 -5 mol), which are good enough to yield a rough idea about the relative stability of self and cross hybridizations.

Validation of DNA melting temperature computation: The DNA melting temperature computation method in SARANI is validated by comparing with the results from the HYTHER DNA Thermodynamics Prediction Server. Scatter Plots of estimated melting temperatures by SARANI and HYTHER show good agreement between the two.