Booster PCR

Booster PCR is an interesting but curiously rarely used PCR technique from the early days of PCR, that aims to increase the sensitivity of a PCR without increasing primer dimers and other non-specific amplification.

The technique, developed by Ruano et al. (1989), involves two PCR steps: a first PCR using very diluted primers to minimise primer dimer formation, followed by a second PCR that uses the first PCR product as a DNA template.

The article where it was first described can be found here:

Ruano et al. (1989). Biphasic amplification of very dilute DNA samples via ‘booster’ PCR. Nucleic Acids Research, 17(13), 5407.

Booster PCR differs from doing a “double PCR” where the first PCR product is used as a template for a second PCR, in that primer concentrations are much lower in the first PCR. Lower primer concentrations are used to reduce the probability of primers coming into contact with other primers, and thereby co-amplifying and producing primer dimers, under conditions of low template DNA concentrations.

Booster PCR also differs from nested PCR in that the same primers are used for both PCRs. This removes the need for additional primers, or the need to reduce the length of amplicons, both of which could be advantageous in some scenarios.

How Booster PCR works

The technique of Booster PCR works as follows:

The first PCR

A first PCR uses primers that are diluted to a concentration of a 10⁷ times molar excess to that of the template DNA. The exact concentrations can be difficult to calculate or may be unknown, so an approximation of 1/10th or 1/5th concentration of standard primer concentrations has been used in some versions.

In this first PCR step, some template amplification should occur, but primer dimers are much less likely to occur because the primers are also low in concentration.

The first PCR will probably not amplify enough DNA to be visible on an electrophoresis gel, but this step should have greatly enriched the PCR product compared to the initial concentration of template DNA.

The second PCR

A second PCR uses a small volume of the PCR product of the first PCR as a DNA template, and uses the same primers at a normal concentration for that PCR application.

In this step, the small amount of target amplicons produced in the first PCR are further amplified to detectable levels as in a normal PCR.

However, because of the initial “boost” of the first PCR, the sensitivity of this PCR should be significantly higher than that of a single PCR.

Variations of Booster PCR

A few authors have added their own modifications to booster PCR, by:

• Using a single-tube approach by adding more primer to the PCR tubes after the first 20 cycles and then continuing for another full set of cycles
• Using a lower concentration of dNTPs in the first PCR, presumably to minimise any additional non-specific amplification
• Using various variations in primer concentration

However, the principle of these variations are essentially the same — an initial PCR boost to the target DNA before the main PCR, using the same primers for both PCRs, but with the primers for the first PCR being very diluted.

By how much can Booster PCR increase PCR sensitivity?

Booster PCR been reported to be up to 10x to 20x as sensitive as a single PCR, and able to detect a single colony-forming unit of Salmonella in a gram of chicken droppings:

Cohen et al. (1994). Detection of Salmonella enteritidis in feces from poultry using booster polymerase chain reaction and oligonucleotide primers specific for all members of the genus Salmonella. Poultry Science, 73(2), 354-357.

The basic procedure has also been modified into a reverse transcription booster PCR where it was found to be up to 100x as sensitive as a single RT-PCR:

De Medici et al. (2004). Reverse transcription-booster PCR for detection of noroviruses in shellfish. Applied and Environmental Microbiology, 70(10), 6329-6332.

Why is Booster PCR so little used?

Booster PCR has been very little used in recent decades, which is curious because it seems at first glance a very good alternative to nested PCR.

We haven’t tried Booster PCR ourselves yet, so we don’t know if there are some other issues that we are missing; if the gains are marginal for most applications; or if nested or hemi-nested PCR are better in terms of reducing PCR artefacts. If you have any ideas, please do let us know!

But if you’re having trouble amplifying an important specimen, and you think it’s due to a low concentration of DNA, it might be worth giving your PCRs a boost with an initial PCR with a 1/10th dilution of primers, then run a normal PCR using the PCR product as a template, and see what happens!

If you give it a try, please get in touch and tell us how it worked for you!