ABSTRACT
The instability of the hybrids is one of the most important factors to be considered when designing oligonucleotide hybridizations. Hybridization and washing conditions are significantly different from those used with long probes. Mismatches substantially reduce the thermal stability of oligonucleotide hybrids particularly for shorter oligonucleotides. Although the stability of oligonucleotide hybrids depends on the concentration of salt, salt concentration is not generally used to control stringency of hybridization. The rate of hybridization is not limiting for single sequence oligonucleotides and in any event the main effect of accelerators is in promoting network formation which will not occur with such short probes. Optimizing conditions is most important for oligonucleotide hybridizations that pilot experiments are first carried out to check and optimize the selected conditions. An alternative means of reducing the complexity of an oligonucleotide pool is to design a single, longer oligonucleotide with an ‘optimized’ sequence.
