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.

1. Applications of
Bioinformatics

2. Insilico
Primer design

3. Primer design
-The most critical parameter for successful PCR
Primer sequence determines
 Length of the product
 Tm
 Ultimately the yield

4. 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

5. 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

6. 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

7. Melting temperature
For oligos in the range of 18-24
Tm
primer
= 2(A+T) +4(G+C)

8. 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

9. Primer design softwares
1. GCG seqWeb
2. Omiga1.1
3. MacVector
4. DNASTAR Primerselect
5. GeneTool PrimerDesigner

14. Restriction enzyme mapping programs

16. Restriction enzyme mapping programs

17. RestrictionMapper

18. RestrictionMapper

19. Microarray analysis