Computer program for calculating the melting temperature of degenerate oligonucleotides used in PCR or hybridization

Degenerate primers or probes have been widely used in molecular cloning, but the calculation of their melting temperatures could not simply be done using thermodynamic parameters because of degeneracy and the lack of a computer program. We present here a simple computer program named dPrimer for the calculation of melting temperature of degenerate oligonucleotides based on the nearest-neighbor model. The program was written in C+2 computer language and implemented in Macintosh with a Symantec C+2 compiler. The degenerate sequencing data were read into a graph data structure. All possible oligonucleotide sequences were then determined by a depth-first search algorithm. Their melting temperature (Tm) values were individually calculated, and output was given as Tm range, mean and standard deviation. These data could help one in the selection of PCR annealing and hybridization temperatures as well as in the design of degenerate oligonucleotides with a desired range of Tm.