R3189 FAQs for NotI-HF, High Fidelity (HF) Restriction Enzymes, Intl, NEB
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NotI-HF FAQ

Q1: What does HF refer to following the name of the restriction enzyme?
Q2: When is star activity a concern?
Q3: Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
Q4: When should I choose the High Fidelity (HF) version of the enzyme?
Q5: Can the change in buffer preference of the HF enzyme be advantageous?
Q6: Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
Q7: What does it mean to be Time-Saver qualified?
Q8: How is the improvement in fidelity of HF restriction endonucleases quantitated?
Q9: What is the Fidelity Index (FI)?
Q10: How does the level of star activity of NotI-HF compare to NotI?
Q11: What is the difference between NotI-HF and NotI?
Q12: What is the specific activity of NotI-HF?
Q13: Is there any difference in the methylation sensitivity between NotI-HF and NotI?
Q14: Is there a difference in cutting close to the ends between NotI-HF and NotI?

Q1: What does HF refer to following the name of the restriction enzyme?

A1: HF stands for high fidelity. Many restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed star activity. HF restriction endonucleases have been engineered to cleave with higher fidelity than the wild type enzyme, hence exhibiting less star activity. Screens using increased glycerol concentration, increased reaction time and high enzyme concentration were used to identify enzymes that would offer the highest fidelity over a wide range of conditions. Learn more about High Fidelity Restriction Enzymes
here.


Q2: When is star activity a concern?

A2: Star activity is of concern if extra banding can cause misinterpretation of results in genotyping and mutational analysis procedures. Experimental design can promote star activity. Small reaction volumes are more likely to contain glycerol concentrations of 5% or greater, a condition known to increase star activity. A 5% glycerol concentration occurs when setting up a double digest in a 20 l reaction using 1 l of each enzyme. Overnight digests are more likely to generate star activity. For tips on avoiding star activity, please
click here.


Q3: Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?

A3: In many cases, changing the charged amino acids of a restriction endonuclease gene results in changes in buffer preference. These changes can be significant. For example, wild type SalI has a strict requirement for NEBuffer 3, a high ionic strength buffer. However, SalI-HF works well in NEBuffer 2 and NEBuffer 4, which are moderate ionic strength buffers.


Q4: When should I choose the High Fidelity (HF) version of the enzyme?

A4: The HF version of the enzyme has the same cleavage specificity as the wild type enzyme, and should be chosen if star activity is a concern or if the recommended buffer is more convenient for double digestion or other multi-step protocol. There is no disadvantage to using the HF version. Learn more about High Fidelity Restriction Enzymes
here.


Q5: Can the change in buffer preference of the HF enzyme be advantageous?

A5: The HF enzymes typically have a different optimal buffer than the wild type enzyme; this can be advantageous when designing double digest experiments. If possible, NEBuffer 4 was chosen as the recommended buffer for an HF enzyme, as NEBuffer 4 shows the highest level of enzyme compatibility in the NEB buffer system. To see a complete list of enzymes that recommend NEBuffer 4 click on
Simplified Buffer System.


Q6: Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?

A6: Star activity was extensively tested in all NEBuffers that showed at least 50% enzyme activity. Star activity is significantly reduced compared to the wild type enzyme activity in all NEBuffers. However, we recommend using the suggested reaction buffer when possible because there are cases where star activity can still be problematic under extreme conditions using non-recommended buffers.


Q7: What does it mean to be Time-Saver qualified?

A7:
Time-Saver qualified enzymes will digest 1g of substrate DNA in 5-15 minutes.


Q8: How is the improvement in fidelity of HF restriction endonucleases quantitated?

A8: Star activity is measured using a Fidelity Index (FI). The FI of the wild type restriction endonuclease can be directly compared to the modified HF version.


Q9: What is the Fidelity Index (FI)?

A9: “The Fidelity Index (FI) is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease.” The FI value translates into the minimum number of units that was required to produce star activity. The FI value can be influenced by substrate, buffer and additives like glycerol.

The following reference explains how the Fidelity Index is determined. Hua Wei et. al. “The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases”, Nucleic Acids Research, 2008, Vol.36, No. 9: e50.


Q10: How does the level of star activity of NotI-HF compare to NotI?

A10: The Fidelity Index which measures the minimum units to produce star activity was determined using the following conditions; 1X NEBuffer, 5% glycerol, substrate pXba (1g/50 l), incubated for 1 hour at 37˚C . The minimum number of units of NotI-HF required to produce star activity in the recommended NEBuffer 4 is (≥64,000 units). The minimum number of units of NotI required to produce star activity in the recommended NEBuffer 3 is (4,000 units).The minimum number of units of NotI required to produce star activity in NEBuffer 4 is (32 units).


Q11: What is the difference between NotI-HF and NotI?

A11: NotI-HF has been engineered to cleave with increased fidelity compared to wild type NotI. The Lysine at position 150 was changed to Alanine (K150A).


Q12: What is the specific activity of NotI-HF?

A12: The specific activity of NotI-HF in the recommended NEBuffer 4 is 5.5 x 105 Units/mg. The specific activity of NotI wild type in the recommended NEBuffer 3 is 9.3 x 105 Units/mg using pXba as the substrate.


Q13: Is there any difference in the methylation sensitivity between NotI-HF and NotI?

A13: Both NotI-HF and NotI perform the same when tested on various CpG methylation patterns. For more specific information regarding methylation, follow the link to the Restriction Enzyme database
REBASE.


Q14: Is there a difference in cutting close to the ends between NotI-HF and NotI?

A14: No. When tested on a series of five oligos (19-23 bases in length) containing the restriction site and an additional 1 to 5 A/T bases from the end, both enzymes cut the oligo with 1 base pair from the end. When designing primers, NEB recommends adding 6 extra bases to ensure against experimental failure due to variation in substrate and reaction conditions.