Minimum Set Primers and Unique Probes Design Algorithms for

Differential Detection of Symptom-Related Pathogens

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Introduction
Methodology
	Tetra-Nucleotide Nucleation (TNN)
	Unique & Common Sections
	Nearest-Neighbor Model
	MCGA
	Linker Design
Computational Results
Bio-Experiment
Conclusion
Reference

The thermodynamic parameters for hybridization between oligo-nucleotides and target sequences are estimated with a nearest-neighbor model. These thermodynamic parameters include melting temperatures ( Tm ), free energy ( Delta G ), enthalpy ( Delta H ), and entropy ( Delta S ). The nearest-neighbor model has been proven to accurately estimate thermodynamic parameters (Rahmann and Grafe, 2004; Tanaka, et al., 2004) . In the nearest-neighbor model, the thermodynamic parameters are calculated from hybridization of consecutive di-nucleotides. For example, the enthalpy will be calculated as follows: (4) where Delta Hnn is the enthalpy of hybridization between two di-nucleotides, and Delta Hinit is the enthalpy for initiation of a DNA duplex. The other thermodynamic parameters (free energy and entropy) are estimated in similar ways. The melting temperature ( Tm ) of annealed sequences are calculated as follows (Sugimoto, et al., 1996) : (5) where Delta H and Delta S are enthalpy and entropy estimated with nearest-neighbor model, R is the gas constant, CT is the molar concentration of the oligonucleotides, and a equals 4. Mismatches are also considered in the calculation of melting temperatures.