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- EnthalpyS- Entropy for helix formationR- Molar gas constantc- 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 contentB/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