Minimum Set Primers and Unique Probes Design Algorithms for

Differential Detection of Symptom-Related Pathogens

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

Though the purposes of unique probes and multiple-use primers are different, common techniques have been used to design the degenerate primers and specific probes. Both primers and probes anneal to target sequences. Therefore many common issues should be considered. The melting temperature of primers/probes need to be calculated (Lockhart, et al., 1996) . Secondary structures of oligonucleotides should be considered, avoiding dimer loops and hairpin loops (Wang and Seed, 2003) . Our algorithm extends the concept of nucleation by exact matches (Wetmur and Davidson, 1968) and uses a hash of tetra-nucleotides as a first-pass filter. The regions on target sequences with least and most Tetra-Nucleotide Nucleation (TNN) sites were identified as unique regions and common regions. Unique probes and multiple-use primers were designed to anneal with these regions, respectively. To reduce the complexity of the primer design, we restricted the length of designed primers to 12 nucleotides. Linker segments (8-mer in length) were added to the 12-mer primers afterward. The linker nucleotide can keep the melting temperatures of primers within a small range. The resulting primers are 20-mer in length, and have melting temperatures around 55°C .