The non-specific binding of primers is always a possibility due to
- sequence duplications;
- mismatches between primer and template;
- partial primer binding where the 5' end does not bind;
- use of too low an annealing temperature;
- use of degenerate primers or primers containing inosine, a modified nucleoside found in tRNA that can pair with the nucleotides A, C and T, also adds to non-specific priming.
When run on an agarose gel, the products of a reaction where there has been non-specific priming may result in either a smear of DNA or many bands. To overcome this problem various approaches can be taken:
- The annealing temperature can be altered;
- The concentration of free magnesium ions (thus stabilising DNA and RNA interactions) can be varied;
- Hot-start polymerases can be used. In this case the enzyme is rendered inactive by blocking the active site with an antibody or chemical which is removed when the reaction reaches 95°C;
- Nested PCR where two sets of primers (outer and inner) are used to amplify the required sequence;
- Touchdown PCR, where the annealing temperature is calculated and then a higher temperature is actually used at the beginning of the PCR. The annealing temperature is then decreased by 1°C every one or two cycles until the calculated temperature is reached.