Expand High Fidelity^PLUS PCR System, dNTPack from Roche Applied Sciences

The Expand High Fidelity^PLUS PCR System, dNTPack from Roche Applied Sciences is a PCR kit which includes all reagents necessary to accurately amplify DNA targets ≤5 kb with high sensitivity: a ready-to-use dNTP mix (10 mM each), 5x reaction buffers (with 7.5 mM MgCl₂, 25 mM MgCl₂ or without MgCl₂) and the unique Expand High Fidelity^PLUS enzyme blend (5 U/µl). Roche Applied Sciences developed this enzyme blend which combines Taq DNA polymerase (5’ - 3’ polymerase activity) and a novel protein that mediates proofreading activity (contains 3’ - 5’ exonuclease activity), but has no polymerase activity itself. The company claims that this new blend of polymerase delivers greater yields and a six-fold higher accuracy than Taq polymerase alone, especially when using small amounts of DNA template. In addition, the Taq in this mix has been engineered to eliminate the need for time-consuming manual hot-start, thereby also limiting contamination due carry-over and evaporation.

In my experience, this is an excellent PCR system which provides accurate and reliable amplifications and is very suitable especially for difficult-to-amplify or single-copy templates. I used the system to amplify genomic DNA of unknown sequence (under 1 kb in size) as well as for establishing a zygosity screening system for transgenic mice. Even though it was difficult to establish the right conditions for the unknown sequence, since I did not know what size to expect, I always obtained single bands in all PCR reactions. To establish the zygosity screening, I tried different conditions. I changed the MgCl₂ concentrations, the amount of template DNA and/or primer. Since the screening included amplifying one or two copies of a gene from genomic DNA of heterozygous or wildtype mice, respectively, as well as several copies from DNA of transgenic mice, I started screening with higher amounts of DNA (30-50 ng). This resulted in no product for the single copy amplification and a weak band for multiple copy reactions. Using less DNA (10 ng-1 ng) and the recommended final concentration for dNTPs (200µM) , 1.5 mM MgCl₂ and 200 nM primer not only improved the yield but also resulted in a cleaner PCR product detected as a sharp band in ethidium bromide-stained gels. The multiple copy reactions were less sensitive to increasing DNA concentrations (up to 30 ng). The PCR reactions always produced a single band (even up to 40 cycles) in comparison with a standard Taq polymerase (without proofreading capability) from another vendor, where I needed not only to increase the DNA amount but also obtained multi-band products. This demonstrates the sensitivity of the enzyme and its success in difficult-to-amplify reactions with limited amounts of DNA. The newly included dNTP mix (dATP, dCTP, dGTP, dTTP), which I had used before in different PCR reactions, is an excellent and reliable mix free of PCR-inhibiting compounds and works well even after many freezing and thawing cycles (up to 50 cycles).

The package insert contains suggestions on how to set up, optimize and troubleshoot PCR reactions with the Expand High Fidelity^PLUS PCR System, dNTPack. There is also some information available on the website about the enzyme and the nucleotide mix, which is also sold separately. In addition, the technical support at Roche Applied Sciences was very helpful in troubleshooting and setting up PCR reactions.

The Expand High Fidelity^PLUS PCR System, dNTPack is a reliable and robust system for accurate amplification especially when amplifying from limited amounts of DNA and/or difficult DNA templates.