Primer Designing- Points to be considered

1. Primer length- while designing primers for normal PCR, primer length should be 18-22 bp.

2. Addition of Restriction Enzyme site with additional bases-

While calculating Tm, No need to consider additional bases or RE cutting site included in primer. RE sites should be added to 5’ end of forward primer and to 3’ end of reverse primer.

3. While calculating Tm value- Primers with Tm 52-58 ⁰C produces better results. Primers with melting temperatures above 65^oC have a tendency for secondary annealing.

The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield. The difference of 5 ^oC or more can lead no amplification.

Nearest neighbor thermodynamic theory-

Formula for primer Tm calculation:

Melting Temperature Tm(^oK)={ΔH/ ΔS + R ln(C)}, Or Melting Temperature Tm(^oC) = {ΔH/ ΔS + R ln(C)} - 273.15

Where ΔH (kcal/mole) : H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in Enthalpy. In the above formula the ΔH is obtained by adding up all the di-nucleotide pairs enthalpy values of each nearest neighbor base pair.

ΔS (kcal/mole) : S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained by adding up all the di-nucleotide pairs entropy values of each nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the default condition used for all calculations.

ΔS (salt correction) = ΔS (1M NaCl )+ 0.368 x N x ln([Na+])

Where

N is the number of nucleotide pairs in the primer ( primer length -1).

[Na+] is salt equivalent in mM.

[Na+] calculation:

[Na+] = Monovalent ion concentration +4 x free Mg2+.

Generally Wallace Rule is used to roughly estimate Tm.

Tm = 4 (G + C) + 2(A + T)

4. GC content- GC content should be 50-60 % in designed primers.

The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.