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Primer design is a critical step in PCR that determines the length, yield, and melting temperature (Tm) of the amplified product. Several variables must be considered when designing primers, including length, Tm, specificity, complementary sequences, GC content, and 3' end sequence. Primer length affects specificity, annealing temperature, and time. Primers between 18-24 bases that have a Tm near 55°C are optimal. Tm can be estimated using formulas considering primer length and GC content. Primer design software tools can help optimize primers while considering these important parameters.

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Applications of Bioinformatics
Insilico Primer design
Primer design -The most critical parameter for successful PCR Primer sequence determines  Length of the product  Tm  Ultimately the yield
Primer design Several variables must be taken into account  Primer length  Melting temperature  Specificity  Complementary Primer Sequences  G/C content and Polypyrimidine (T,C) or Polypurine (A,G) stretches  3’-end Sequence
Primer length Primer length determines Specificity Temperature and Time of annealing Oligonts b/w 18 and 24 bases are extremely sequence specific When aiming for an annealing temperature of at least 50°C, this corresponds to a primer with a calculated Tm ~ 55°C
Melting temperature Both of the oligonuleotide primers should have similar Tm Melting temperature of oligoprimer Tm primer = ∆H[∆S + R ln (c/4)] – 273.15°C + 16.6 log10[k+ ] H- Enthalpy S- Entropy for helix formation R- Molar gas constant c- Concentration of Primer
Melting temperature For oligos in the range of 18-24 Tm primer = 2(A+T) +4(G+C)
Other parameters Specificity: Primers must have a unique sequence within the template DNA Complementary sequences: There should be no intra-primer homology beyond 3 base pairs G/C content B/W 45% to 55% GC
Primer design softwares 1. GCG seqWeb 2. Omiga1.1 3. MacVector 4. DNASTAR Primerselect 5. GeneTool PrimerDesigner
Restriction enzyme mapping programs
Restriction enzyme mapping programs
RestrictionMapper
RestrictionMapper
Microarray analysis