The definition of FI
An example of a typical gel from an FI measurement experiment is shown in . λ DNA is cleaved by ScaI in buffer N3. Each lane contains the same amount of DNA (0.6 μg unless stated otherwise). The amount of enzyme is reduced as a 2-fold serial dilution across the gel. At the far right, there is very little enzyme, so little or no DNA is digested. As the amount increases toward the left, partially digested fragments start to appear, which eventually become fully digested when the amount of the enzyme reaches a critical point. This we call the lowest REase amount needed for complete cleavage on cognate sites (LCC). At this point, all bands that can only be generated by this specific REase cleaving at its designated recognition sequence reach their highest intensities, and no other bands are present. This gives rise to the unique cleavage pattern that can be predicted based on the DNA sequence of the substrate and the recognition sequence of the enzyme. This pattern remains as the REase amount continues increasing until the star activity of the enzyme begins to appear. The amount of the REase for the lane that immediately precedes the initial star activity lane will be considered the highest amount showing no star activity (HNS). Once star activity appears, the normal cleavage band(s) is further cleaved into smaller fragments, visible as a weakening of the normal band(s) and the appearance of a new star activity band(s).
Figure 1. Definition of fidelity index. λ DNA is cleaved by ScaI in buffer N3. The enzyme concentration is reduced by a series of two fold dilutions in diluent A. Lane1: 0.5 μg NEB 1-kb DNA Marker; lane 2 contains 600 U; lane 10 contains 2.4 U; (more ...)
We define the FI, which describes the degree of star activity for a REase, as the ratio of the highest REase amount showing no star activity (HNS) to the lowest REase amount needed for completed cleavage on cognate sites (LCC):
For the above example, the LCC is 0.6 U, because that is the minimum amount of enzyme needed to digest 0.6 μg DNA in 1 h. The HNS is 2.4 U because above that amount additional bands begin to appear due to star activity. Thus the FI of ScaI in N3 is 2.4/0.6 = 4. The FI is an indicator of how specific an enzyme is. The higher the number, the greater the specificity of the enzyme and the larger the window of enzyme concentration that can be used without the deleterious consequences of star activity.
The FIs for selected REases
Because an accurate determination of the FI often requires high enzyme concentration, only 213 enzymes were available at suitable concentrations. The FIs of these 213 REases in four buffers (N1–N4) were measured. The FIs for the 56 most representative REases are listed in . The comparison of the LCC lanes in each buffer also indicates the relative activity of the REase in various buffers. The relative activity in the best buffer is designated as 100%, and those in the other buffers are the relative activity percentage numbers. It should be emphasized that the FIs listed in are all determined under normal reaction conditions, i.e. DNA was at 20 ng/μl, REase was at 10% of the volume and reaction was at the recommended temperature for 1 h. Compared to the conditions for the activity unit determination, the conditions for the FI determination are the same except for the selection of substrate in some cases. When there are too many bands (such as the 4-base REase MspI on λ DNA) the extra DNA bands caused by star activity can be difficult to observe. On the other hand, when the substrate is relatively short, such as when the small plasmid pNEB193 is used as the substrate in the unit definition for the 8-base REase PacI, the sequence complexity of the substrate may be too low to contain star activity sites, so that star activity cannot be detected. In both cases, a suitable substrate of appropriate size was selected to demonstrate both cognate activity and star activity.
Selected list of Fidelity Indices of restriction endonucleases
Because of the way in which the FI determination assay was designed, all FIs have values of 2n, with n being a positive or negative integer. For convenience, the FIs were recorded as 0.02, 0.06, 0.13, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 120, 250, 500, 1000, 2000, 4000, 8000, 16 000, 32 000, 64 000. FIs for different REases in different buffers varied from as small as 0.02 (ScaI in buffer N1) to as large as ≥64 000 (HaeIII in buffer N2). When no star activity was seen even at the highest enzyme concentration tested, a greater-than-or-equal-to number is given.
FIs of 500 and above (A) indicate that the REase can be used with as much as a 500-fold overdigestion in that buffer. An FI of 120 or 250 (B) indicates that there is a good range of REase concentrations that do not lead to visible star activity while the reaction is still complete. An FI of 64 or less suggests that caution should be used because the REase may show apparent star activity in the tested buffer. Often digestions are allowed to proceed for more than 1 h, which will worsen the situation if star activity is present. In some cases, the FI can be <1, which means that the star activity bands appear even before the normal complete digestion pattern is established.
Figure 2. Representative examples of the ‘Excellent’ and ‘Good’ classes of REases in terms of star activity. (A) REase in the ‘Excellent’ class. Cleavage of pBR322 by MspI in buffer N1: MspI is 2-fold serially diluted (more ...)
Some FIs require special consideration. For instance, a low FI may be caused by a rather low relative activity at canonical sites, such as AscI in buffer N1. Here, the FI is 8 but the activity relative to the best buffer is only 0.1% and as a result is labeled as 0%. Some REases also cannot complete the reaction at all in certain buffers, no matter how much is added; here the FI is listed as NC (Not Complete). Practically speaking, an REase will never be recommended for use in a buffer in either of these two cases. Some FIs are inconclusive numbers, which are greater than or equal to a number that is no more than 64, such as ≥1, ≥2, ≥4, ≥8, ≥16, ≥32, ≥64. The actual FI can be high yet can also be low. This happens because the available REase concentration is too low to find the concentration that exhibits star activity. This can only be resolved when a higher starting concentration of the REase becomes available. The FIs in the above three cases are not used for the further classification as described below.
Based on the FIs in all four tested buffers, the enzymes can be categorized into four different classes depending on their different behaviors in the tested buffers. The four classes are described below.
At least one FI is at least 500 and all FIs are at least 120. For this class, there is at least one buffer in which little or no star activity is present and there is no buffer in which severe star activity is seen. Eighty-three REases fall into this class. The cleavage pattern of one member of this class, MspI, is shown in A. No star activity was seen even at the highest REase amount (9600 U).
All FIs are either 120 or 250. Here at least one buffer can be used without having significant star activity, and still there is no buffer that will cause severe star activity. Twenty-two REases are in this class. A representative example (BstBI) is shown in B.
At least one FI is no more than 64 and no FI is more than 250. The enzymes from this class have traditionally been considered to display star activity (ScaI, ). The basic reason why this cut-off range at 250 is selected is that it applies to the ‘classic’ example of star activity, EcoRI, which has an FI of 250 in buffers N1 and N3 and an FI of 4 in buffers N2 and N4. Ninety-eight of the studied enzymes belong to this Class.
At least one FI is ≥500, while at least one FI is no more than 64. These REases can be used in some buffers with little or no star activity and in another buffer with significant star activity. Ten REases are of this type (NotI, ).
Figure 3. Representative example of the ‘Variable’ class of REase. Comparison of FIs of NotI in buffer N4 (A) and buffer N3 (B) using pXba DNA as the substrate. NotI is 2-fold serially diluted with diluent C. The reaction contains 3 μl of (more ...)
The FI of REases is independent of the supplier
The same REases from different commercial suppliers were compared. EcoRI and BamHI from eight different companies as listed in the ‘Materials and methods’ section were all tested in the same buffers N2 and N4. These two REases were selected because not only are they well-known REases with star activity, but also that they are readily available at concentrations sufficient to show a range of star activity. Since the FIs comprise a set of values of 2n, the designation of the partial activity band and the star activity band could be off by one lane depending on the starting REase concentration. So a variation of 2- to 4-fold is considered to be identical within experimental error. The FIs were essentially the same for EcoRI and BamHI from different suppliers (). Additionally, HindIII, NotI and PstI from Fermentas International Inc. were also checked in buffers N2 and N4, and the values were found to be similar to ones from NEB. While it would be desirable to test every REase from each REase suppliers, the availability of high concentration REases from suppliers other than NEB is limited. Our limited tests confirm that star activity is an intrinsic property of the REase and is independent of supplier. However, because the FI can vary a lot in the different buffers, the exact FI in the buffer system from other suppliers remains to be determined.
FI variation from different commercial sources
Star activity is site specific
While it has been shown by many groups that star sites for a few enzymes are different from nonspecific sites, i.e. star sites have a certain specificity and are not random (12–14
), this is the first time that star activity has been studied on such a large scale. Since star activity results from cleavage at specific secondary sites and is observed as extra discrete and unique band(s) other than the ones from the cognate sites, a cautionary note is necessary. This is illustrated in for NotI. While clear star activity bands were seen when pXba (22 kb) was used as the substrate, no star activity was seen with pBC4 (11 kb, a subset of pXba). The reason is that the major star activity site of NotI (ACGGCCGC) (50
) is present in pXba but not in pBC4. Theoretically, for star activity, one positive result is sufficient to prove the REase has star activity, while even many negative results from different substrates can only suggest that the tested REase has no apparent star activity.
Figure 4. Star activity is site-specific. While a clear star activity band is seen for NotI when pXba is used as the substrate, no star activity is observed when pBC4 is used. Asterisk is a star activity band, and hash is a partial activity band in every panel. (more ...)
FI varies in different reaction buffers
Just like the normal cleavage activity of a REase, its star activity also varies from one buffer to another. Enzymes of the Variable class in are extreme cases where there could be as great as a 2000-fold difference for FIs in different buffers. There are also a significant number of enzymes that have very similar FIs in all four different buffers. For all the enzymes studied, the percentages of enzymes that show the highest FIs in N1, N2, N3 and N4 are 24, 24, 24 and 28%, respectively. So generally speaking, there is no buffer that is much better than the others in terms of giving much less star activity. This is in total contrast to the normal cleavage activity of REases, where 41% of the REases are most active in buffer N4, followed by 25% in N2, 20% in N1 and 14% in N3. REases are ideally used in their optimal buffers; however, restriction digestions that include two or more REases with different optimal buffers are very common. This is why a selection of four reaction buffers instead of one was tested in this study.
Re-classification of the REase class
Through this study, we have found some REases which have previously been flagged as having star activity to actually be in the ‘Excellent’ or ‘Good’ Classes of REases, and vice versa. Enzymes of the former group include AvaI, BsoBI, BssHII, BstZ17I, HpyCh4III, NciI, PflFI, TfiI, XmnI and ZraI. The reason for the earlier categorization of these enzymes as possessing star activity is probably because the previous testing conditions are somewhat different from this study, such as with higher glycerol or longer incubation times. Fifty-seven REases, such as AgeI, previously considered as very specific are now shown to have star activity. Insufficient investigation had previously failed to detect and confirm the star activity of these REases.
FI variation on different substrates
It is well known that REases cleave different substrates with varying efficiency, because each individual substrate will have a different arrangement of REase sites, and each site will lie in a different sequence context which is expected to affect the digestion rate. Since the star activity is also a cleavage activity with specificity, the star activity will also vary on different substrates and will lead to unique star activity band(s). Thus, we expect that the FI will vary on different substrates.
For BamHI, different substrates gave different FIs in the same buffer N2. While BamHI has the cognate site, GGATCC, one group of major star sites for BamHI is GGNTCC (19
) under normal reaction conditions, which can be greatly enhanced in the presence of glycerol. pMAL-c2E and pGPS3 had an FI of 1, while many substrates gave an FI of 2, such as pTWIN-2, pUC19, pTWIN-MBP1 and LITMUS28i. The FIs for λ DNA, pBR322 and pXba are 4. pBC4 had the highest FI of 8.
From this example of BamHI, an FI can vary up to 10-fold, even higher in some extreme cases. Although the FIs for different REases are measured on different substrates, the general trend is maintained, i.e., for a group of substrates, an enzyme from the ‘Excellent’ class is normally better than an enzyme from the ‘Star-prone’ class.
FI varies with reaction temperature
The digestion of λ DNA by BstEII was studied at different temperatures. The FIs for 25, 30, 37 and 60°C are ≥64, 32, 32 and 16, respectively. It was found that while a higher temperature slightly increased the normal cleavage activity of this enzyme, it increased the star activity to a larger extent. This was responsible for the decreased FI at the higher temperatures. Thus it is probably advisable to use temperatures in the lower range to ensure fidelity of cleavage and minimize star activity. BsoBI, a REase which has higher activity at 65°C, is most appropriately used at 37°C to minimize the star activity. The FIs of BamHI and OkrAI at room temperature have been found to be about 10 times higher than that at 37°C (Xu,S.-Y., unpublished data).
Effects of additional external factors
It has been shown experimentally that the star activity of a REase can be enhanced by additional factors such as organic solvents or unusual metal ions (18–20
). Since the FI is a quantitative measurement of star activity, it is also altered by these additional factors. The FIs of PstI in buffer N3 on λ DNA, with different additional factors, are listed in . Increased amount of glycerol, DMSO, ethanol or MnCl2
all reduce the FI significantly.
The effects of additives on the FI of PstI
REases have the same FI on supercoiled and linearized DNA
Some DNAs are typically available in supercoiled form, while others are usually in linear form. We tested whether the differences between these two forms had an effect on the FI. The FIs of BamHI, EcoRI, PvuII, SphI and SspI on supercoiled pBR322 and PstI-linearized pBR322 were compared and found to be the same. The REase amounts needed for the HNS and the LCC are also essentially the same (data not shown).
FI and relative activity varies much in the PCR extension mixture
The FIs and relative activities of 16 REases have been determined in two PCR extension mixtures. The data are listed in . The two PCR extension mixtures represent two different buffer conditions, which have a higher pH than normal reaction buffers. Some REases perform well in the two PCR extension mixtures, such as AatII, HaeIII, HindIII, MseI, NheI and XbaI. Others either have much less activity such as BglII, NdeI or a much lower FI such as BamHI, EcoRI, NcoI, NotI, SmaI, SpeI and SphI. Occasionally, both relative activity and FI are less than in the normal reaction buffer. BsrGI is one such example. Unless a REase is specifically assayed in a PCR extension mixture, it seems unwise to assume that the FI will not be affected.
FIs in the PCR extension mixture