Expand High Fidelity Plus PCR System From Roche Applied Science

The Roche Expand High Fidelity^PLUS PCR System contains a master mix enzyme blend of Taq DNA polymerase and a proofreading enzyme that lacks polymerase activity. These enzymes are designed to increase yield and fidelity when amplifying fragments up to 5 kb. The system claims to have a 2-fold increase in fidelity over the Expand High Fidelity PCR System and a 6-fold increase in fidelity compared to Taq DNA polymerase alone. This novel proofreading protein was isolated and characterized by Roche Applied Sciences and little information was given about it. This system also uses the dUTP incorporation method to prevent PCR cross-contamination, if needed. Each picoliter of PCR product can contain 10,000 molecules; therefore simple aerosolization can lead to contamination of subsequent reactions even in the cleanest of environments. To prevent this, dUTP is incorporated instead of dTTP during elongation. This must be done in all PCR reactions to adequately prevent contamination. This system uses a 20 minute pre-PCR incubation with Uracil-DNA-Glycosylase to selectively destroy DNA containing dUTP that would have been carried over from a previous reaction. In addition, the system can be used for labeling DNA fragments with radioactive and non-radioactive modified nucleotides.

The Expand High Fidelity Plus PCR System comes wit the Expand High Fidelity Plus enzyme blend at 5 U/ul; the 5X reaction buffer contains 7.5 mM MgCl₂, 5X reaction buffer without MgCl₂, and a 25 mM MgCl₂ stock solution. The Roche Applied Sciences website contains great information on this product; the system also comes with an informative and detailed manual.

This PCR system is used just like all other PCR kits. The difference is only the proprietary proofreading enzyme in the Taq master mix. The system recommends a starting MgCl₂ concentration of 1.5 – 4 mM if using the reaction buffer without MgCl₂. However, I tend to start with the reaction buffer containing 7.5 mM and optimize from there. They also recommend using 2.5 U of the enzyme blend per reaction. The manual gives the standard reaction procedure as well as the optional procedure for preventing carry-over: Simply add 2 U of Uracil-DNA Glycosylase to the reaction as well as 600 uM dUTP along with the standard dNTP mix. This system does not supply the dNTP mix, but Roche carries a PCR nucleotide pre-mix that works well.

I have used this kit for many PCR reactions and had excellent luck with all of them. This worked particularly well for amplification of small fragments from small amounts of plasmid DNA. I had trouble getting a clean fragment from a particular reaction using standard Taq polymerase mixes. The Expand High Fidelity Plus PCR System enabled me to get a clean fragment on the first try. I also like this system because it does not require a hot start, which lessens the amount of time required to complete the PCR reaction. The main problem I have had with this kit is the 5kb size restriction. Occasionally, I do need to PCR large fragments and this kit does seem to do much better with smaller fragments. I have noticed reduced efficiency with fragments over 3kb. In my experiments, the smaller fragments of 1.9 kb down to 150 bp have 3-4 times greater concentration, on average, than the fragments of 3.2 and 3.9 kb that I have tried to amplify.

Overall, I have been very satisfied with this kit. I do a lot of difficult PCR reactions due to low copy plasmids in Staphylococcus aureus which is notorious for being difficult to lyse. In order to get adequate concentrations, my reactions usually require optimization of annealing temperature, elongation time and primer/DNA concentrations. This kit has taken away a lot of the guesswork and made optimizing the PCR reactions easier and quicker. In addition, sequencing has shown the enzyme fidelity to be extremely high; we have been able to detect single bp deletions.

Katrina Stumpf
Senior Lab Specialist
McGuire VA Medical Center
Division of Infectious Disease