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High Fidelity (HF®) Restriction Enzymes

The Latest Innovation in Restriction Enzyme Technology

New England Biolabs provides customers with high quality tools for a wide range of molecular biology applications. As part of our ongoing commitment to the study and improvement of restriction enzymes, we are pleased to introduce a line of High Fidelity (HF) restriction enzymes. These engineered enzymes have the same specificity as their established counterparts. However, certain properties have been altered, including buffer requirements and enzyme fidelity. These modifications provide customers with more flexibility in setting up their restriction enzyme digests. The overall goal of engineering restriction enzymes is to provide improved enzymes that will allow more flexibility with respect to reaction volume, incubation time and buffer compatibility. Each of these enzymes has been purified to the same high standards as our other restriction enzymes, and are available at the same low price.

The introductory selection of engineered restriction enzymes offers the benefit of reduced star activity. Star activity, or relaxed specificity, is an intrinsic property of restriction enzymes. Most restriction enzymes will not exhibit star activity when used under the recommended reaction conditions. However, for enzymes that have reported star activity, extra caution must be taken to set up reactions under the recommended conditions to avoid unwanted cleavage (More information on Star activity). Different techniques such as cloning, genotyping, mutational analysis, mapping, probe preparation, sequencing and methylation detection employ a wide range of reaction conditions and require the use of enzymes under suboptimal conditions. These new high fidelity (HF) enzymes will offer increased flexibility to reaction setup, maximizing results under a wider range of conditions.

ScaI is one of the most frequently reported enzymes to exhibit star activity (unwanted cleavage). While star activity is observed with ScaI in NEBuffer 3 (supplied buffer) as well as NEBuffer 4, star activity is significantly reduced with ScaI-HF. 20 l reactions were set up containing 2 l of enzyme and incubated for 1 hour. Conditions were designed to simulate those obtained when a double digest is performed in a 20 l reaction. Marker M is the 1 kb DNA Ladder (NEB #N3232).

EcoRI from various suppliers produces the correct banding pattern when 10 units are used, however, star activity is observed with larger amounts of enzyme. Star activity is not observed with EcoRI-HF, even at higher enzyme amounts. Reactions were set up according to recommended reaction conditions of each manufacturer. Reactions contained 1 g Lambda DNA in a 50 l reaction volume and were incubated overnight at 37C. Marker M is the 1 kb DNA Ladder (NEB #N3232).

In addition to reduced star activity, all of these engineered enzymes work optimally in NEBuffer 4, which has the the highest level of enzymes compatibility, and may simplify double digest reactions. They are also Time-Saver qualified, and will digest substrate DNA in five minutes.

In order to distinguish these engineered enzymes, the letters HF® have been added to the restriction enzyme name. An icon designating that the enzyme has been engineered will appear with the product entry, on the datacard and on the website. In addition, icons for the enhanced properties that these new enzymes possess will also be included. These enzymes will be packaged with a purple cap to distinguish them from our existing restriction enzymes.

The following HF enzymes are currently available:

AgeI-HF® EcoRI-HF®
BmtI-HF® HindIII-HF®
BsaI-HF® KpnI-HF®


Please check back frequently to learn more about this growing list of restriction enzymes from New England Biolabs!

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