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How to Design Primers for PCR and quantitative real time PCR (qPCR) Tips for primer design:

Keep the melting temperatures (Tm) of each primer pair within 2?C of one another.Cytosine and guanine have stronger binding affinity than adenine and thymine and repeats of more than 4 G or C can bind to many places in the genome with high affinity.A GC content between 35% and 65% without long stretches (> 4 bases) of the same nucleotide will ensure enough sequence complexity for optimal primer specificity.Amplicons between 70-140 base pairs are generally long enough to allow the design of two efficient primers and a probe (if a TaqMan-based assay is desired) for qPCR assays.Having a similar Tm between primers ensures that the forward and reverse primers will be bound to their complementary DNA strands at the same time, reducing the chance that the primer with the highest Tm will bind to nonspecific DNA sequences.If these repeats are at the 3' end of the primer, DNA polymerase can extend amplicons in off-target locations, which can ultimately decrease the PCR efficiency.

Original text

How to Design Primers for PCR and quantitative real time PCR (qPCR)
Tips for primer design:


Keep the melting temperatures (Tm) of each primer pair within 2°C of one another. The Tm can be approximately calculated by the formula Tm = (A+T) x 2 + (G+C) x 4, however, more precise, and elaborate Tm calculation tools are available online. Having a similar Tm between primers ensures that the forward and reverse primers will be bound to their complementary DNA strands at the same time, reducing the chance that the primer with the highest Tm will bind to nonspecific DNA sequences.
Use an annealing temperature (Ta) 3-5°C below the melting temperature. The annealing temperature is the temperature at which the primers will bind to a new template strand. This needs to be lower than the Tm so that the primers can efficiently bind to the target, but not too low that the primers bind to nonspecific targets.
Keep primers between 18-22 base pairs long. This primer length is long enough to ensure binding specificity while also short enough to keep the Tm within an appropriate range.
Design primers with a GC content of 35-65%. A GC content between 35% and 65% without long stretches (> 4 bases) of the same nucleotide will ensure enough sequence complexity for optimal primer specificity.
Minimize G/C repeats, especially at the 3’ end of the primer. Cytosine and guanine have stronger binding affinity than adenine and thymine and repeats of more than 4 G or C can bind to many places in the genome with high affinity. If these repeats are at the 3’ end of the primer, DNA polymerase can extend amplicons in off-target locations, which can ultimately decrease the PCR efficiency.
Limit amplicon length to 140 base pairs. Amplicons between 70-140 base pairs are generally long enough to allow the design of two efficient primers and a probe (if a TaqMan-based assay is desired) for qPCR assays. Longer amplicons are possible, although it may be necessary to adjust the thermal cycler protocol to allow for complete elongation of the new strand. Additionally, if the sample contains heavily fragmented DNA, it is best to keep amplicons smaller to increase the likelihood of having intact target sequences for primer binding.


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