Question

Give the importance of the following in the context of PCR: (1) melting temperature (Tm), (2) GC content, (3) primer size, and (4) primer specificity

Answer 1

1. Melting temperature:

In PCR, during the annealing phase, the reaction temperature needs to be sufficiently low to allow both forward and reverse primers to bind to the template but not so low which reduce reaction efficiency.

Primers with melting temperature 52-58 degrees centigrade generally produce the best results. The melting temperatures above 65-degree centigrade have a tendency for secondary annealing.

All primers in the reaction should have a similar melting temperature so they anneal to and dissociate from the complementary DNA sequences at approximately the same temperatures, allowing each amplification to precede at the selected temperature.

2. GC content:

The GC content of the sequence gives a fair indication of the primer melting point. A higher GC content indicates a relatively higher melting temperature.

GC bonds contribute more to stability. Increased melting temperatures of primer and template, binding more than AT bonds.

3. Primer size:

The shorter the primers, the more efficiently they can anneal to target DNA. For ideal amplification, the best primers are 17 to 24 bases long. Primers that are longer- say 28 to 35 bases - work better for troubleshooting closely related species. The longer range allows for higher specificity and room for adding restriction enzyme sites to the primer end, if cloning.

Short primers are mainly used for amplifying a small, simple fragment of DNA. On the other hand, a long primer is used to amplify a eukaryotic genomic DNA sample.

4. Primer specificity:

Good PCR primers strike a fine balance between specificity and amplification efficiency. Specificity is controlled by primer length and annealing temperature.

The ratio of primer to the template is also important regarding the specificity of PCR. If the ratio is too high, PCR is more prone to generate unspecific amplification products, and also primer-dimers are formed.