Primer Dimers Real Time PCR

Primer Dimers Real Time PCR.

Real Time PCR Primer Dimers

Primer dimers are short PCR products formed when PCR extension occurs on primers that have interacted with each other. Primer dimers must always be shorter than the amplicon and will therefore have a lower melting temperature than the authentic target. The peak is often wider that the target peak since different combinations of primer/primer interactions can result in a variety of short products with slightly different melting points. The likelihood of their forming during amplification will be determined by the simple competitive binding kinetics of primer/primer and primer/template interactions.

A high primer concentration and low target template concentration will therefore favour primer dimer formation, but overall the design of the primers is the strongest determinant. Primers with runs of complimentary bases, particularly at the 3' end, will have a strong tendency to dimerise but this can be minimised during primer design. In a well designed assay, primer dimers will not form at all, or will only reach detectable levels at very late cycles (CT>37).

The potential for primers to form dimers is most easily tested by running a 'no template' reaction along side a positive cDNA reaction. Fig.2 shows the perfect result - a single peak in the cDNA reaction and no primer dimers even in the absence of template. Fig.3 has a single peak in the cDNA column but the no template control shows that the primers do have potential to interact. If the target gene is abundant and the dimers only form late in the reaction then the assay may still be sound but further analysis is required. Fig.4 shows a very poor assay - even with template present, dimers are still forming (as seen by the second peak) and in the no template control there is massive dimer formation.

---

Related Items:

• Melting Curves Real Time PCR
• Primer Dimer Effect Real Time PCR
• Real Time PCR
• Real Time PCR mRNA Quantitation
• Standard Curves Real Time PCR