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

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 ( Ta) 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 (Tm) of primers/oligonucleotides which enables us to get Tm for single sequence/ input at a time. Hence, we developed an MS-Excel based programme to calculate Tm 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. Keywords: PCR, DNA based markers, Marker Assisted Selection (MAS,) Melting Temperature (Tm), Annealing Temperature (Ta), MS-Excel I Introduction One of the major concerns of modern agriculture is the identification and utilization of valuable genes of agronomic importance in crop plants. The development and use of DNA based molecular markers to track loci that are tightly linked to important genes have irrevocably changed the fields of genetics and plant breeding. Molecular markers exhibit a high degree of polymorphism and simple inheritance patterns with minimal influence of environment and epistasis. Therefore, in combination with conventional breeding approaches, DNA based molecular markers can be used to monitor the presence or lack of these genes in breeding populations and monitoring the level of gene expression under biotic and abiotic stress situation through Marker Assisted Selection (MAS). Several techniques have been developed for the past few years for distinguishing the individuals at DNA sequence level. Most of the markers are PCR (Polymerase Chain Reaction) based and are individually amplified by using oligonucleotide primers. If there is no prior sequence information available in the database for a particular species, random markers like Random Amplified polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP) and Inter Simple Sequence Repeats (ISSR) can be employed. These are multilocus, mostly dominant genetic markers usually produce multiple DNA fragments (each of which is considered as a locus) allowing the generation of a large number of loci across the genome without prior genome sequence information. On the other hand, if sequence information is available, then species specific markers like microsatellites or Simple Sequence Repeats (SSRs) and Single nucleotide polymorphisms (SNPs) can be used for genotyping. In all the PCR based markers, PCR will be efficient under optimal conditions like template DNA concentration, salt concentrations and primer annealing temperature (Ta). Keeping other parameters in PCR constant , the successful amplification of a particular locus depends on optimum Ta which in turn depends on primer sequence and GC content, complementarities of forward and reverse primers, reaction volume etc. The primers of different markers vary in length and sequence composition (GC content) which in turn leads to the variation in primer annealing temperature (Ta). Primer annealing is a physical process where the primers will jiggle around and hydrogen bonds will be constantly formed and broken between specific bases in the single stranded primers and the single stranded template DNA. More stable bonds last a little bit longer (primers that fit exactly) and on that little double-stranded piece the DNA polymerase can attach and start copying the template and thereby results in doubling target sequence.