Computation of Melting Temperatures (Tm) of oligonucleotides for high throughput PCR using MS-Excel

Abstract

DNA based molecular markers have enormous potential to improve the efficiency and precision of conventional plant breeding via marker-assisted selection. Most of the molecular markers are individually amplified by oligonucleotide primers using PCR. The efficiency of PCR depends on primer annealing temperature (T a) which optimize amplification and minimize non-specific binding of primers to the template. In contrast to the majority of the software programs available to calculate melting temperature (T m) of primers/oligonucleotides which enables us to get T m for single sequence/ input at a time. Hence, we developed an MS-Excel based programme to calculate T m for a large set of primers with one click based on two formulae viz., basic and salt adjusted depending on the size of the primers. A wide range of oligonucleotides with size range between 10 to 30mer covering RAPD, AFLP, ISSR SSR and SNP markers were tested which are widely used for marker-assisted selection.