2.5.2 Primer Selection

Unfortunately, the approach to the selection of efficient and specific primers remains somewhat empirical. There is no set of rules that will ensure the synthesis of an effective pair of primers. Yet it is the primers that more than anything else determine the success or failure of an amplification reaction. The following guidelines aid primer design:

1. Whenever possible, select primers with a random base distribution and a GC content similar to that of the amplified fragment.
2. Avoid sequences with a significant secondary structure (e.g., hairpin loops), particularly at the 3’ end of the primer.
3. Check the pair of primers for mutual complementary. In particular, avoiding primers with 3’ overlaps will reduce incidence of primer dimer formation.

Most primers will be between 18 and 25 bases in length and the optimal amount to use in an amplification will vary. Sequences not complementary to the template can be added to the 5’ end of the primers. These exogenous sequences become incorporated into the double-stranded PCR product and provide a means of introducing restriction sites [125] or regulatory elements at the ends of the amplified target sequence [126].

Primer dimer is an amplification artefact often observed in the PCR product, especially when many cycles of amplification are performed on a sample containing very few initial copies of template. It is a double-stranded fragment whose length is very close to the sum of the two primers and appears to occur when one primer is extended by the polymerase over the other primer. The resulting concatenation is an extremely efficient PCR template that can, if it occurs at an early cycle, easily overwhelm a reaction and become the predominant product.

All primers used in this thesis were designed using the Oligo programme¹³ (version 4.1) by Rychlik  [127].

© 2001 Alexander Binder