《英语教学》（英文版）9 Restriction Endonucleases

with l ug of a mixture of single-and double-stranded, H-labeled E. coli DNA (200,000 cpm/ug) in a 50ul reaction volume with the supplied buffer Incubations(along with a background control containing no enzyme) are at the recommended temperature for four hours. Exonuclease contamination is indicated by the percent of the total labeled dna in the reaction that has been rendered TCA-soluble. The limit of detectability of this assay is approxi mately 0.05%. Enzymes showing background levels of degrada tion with 100 units incubated for four hours can be considered virtually free of exonuclease Ligation/Recut Assay Ligation and recutting is a direct determination of the integrity of the DNA fragment termini upon treatment with the restriction enzyme preparation. Ligation and recut of greater than 90% with a 10-to 20-fold excess of enzyme creating ends with overhangs or 60% for blunt ends indicate an enzyme virtually free of exon clease or phosphatase specific for the overhang being tested Alternative assays (i.e, end-labeling) are used to evaluate Type IIS restriction enzymes(e.g, Fokl, Boll). Since these enzymes cleave outside of their recognition sequence, the standard ligation assay would not determine a loss of terminal nucleotides due to exonuclease. The resulting ends could still ligate, and since their recognition sites remain intact, the enzyme would still be able to Blue-White Screening Assay The B-galactosidase blue-white selection system is also applied to determine the integrity of the DNA ends produced after diges- tion with an excess of enzyme to test ligation efficiency. An intact gene gives rise to a blue colony; while an interrupted gene, which contains a deletion due to degraded DNA termini, gives rise to a white colony. Restriction enzymes tested using this assay should produce fewer than 3% white colonies. The values given for the number of units added giving virtually contaminant-free"preparations are somewhat arbi trary. They are useful, however, for determining maximum levels of enzyme to use in a reaction for most common applica tions. Enzymes with quality control results significantly below these values can still be used with confidence under simple assay conditions. as discussed later for complex restriction digestions, caution should be considered when extending reaction times and adding more than 1 to 2ul of enzyme to 1 ug dnA in 50ul Restriction Endonucleases

with 1mg of a mixture of single- and double-stranded, 3 H-labeled E. coli DNA (200,000 cpm/mg) in a 50ml reaction volume with the supplied buffer. Incubations (along with a background control containing no enzyme) are at the recommended temperature for four hours. Exonuclease contamination is indicated by the percent of the total labeled DNA in the reaction that has been rendered TCA-soluble. The limit of detectability of this assay is approxi￾mately 0.05%. Enzymes showing background levels of degrada￾tion with 100 units incubated for four hours can be considered virtually free of exonuclease. Ligation/Recut Assay Ligation and recutting is a direct determination of the integrity of the DNA fragment termini upon treatment with the restriction enzyme preparation. Ligation and recut of greater than 90% with a 10- to 20-fold excess of enzyme creating ends with overhangs or 80% for blunt ends indicate an enzyme virtually free of exonu￾clease or phosphatase specific for the overhang being tested. Alternative assays (i.e., end-labeling) are used to evaluate Type IIS restriction enzymes (e.g., FokI, MboII). Since these enzymes cleave outside of their recognition sequence, the standard ligation assay would not determine a loss of terminal nucleotides due to exonuclease. The resulting ends could still ligate, and since their recognition sites remain intact, the enzyme would still be able to recut. Blue-White Screening Assay The b-galactosidase blue-white selection system is also applied to determine the integrity of the DNA ends produced after diges￾tion with an excess of enzyme to test ligation efficiency. An intact gene gives rise to a blue colony; while an interrupted gene, which contains a deletion due to degraded DNA termini, gives rise to a white colony. Restriction enzymes tested using this assay should produce fewer than 3% white colonies. The values given for the number of units added giving “virtually contaminant-free” preparations are somewhat arbi￾trary. They are useful, however, for determining maximum levels of enzyme to use in a reaction for most common applica￾tions. Enzymes with quality control results significantly below these values can still be used with confidence under simple assay conditions. As discussed later for complex restriction digestions, caution should be considered when extending reaction times and adding more than 1 to 2ml of enzyme to 1mg DNA in 50ml. Restriction Endonucleases 235

How Stable Are Restriction Enzymes? As a class, most restriction enzymes are stable proteins. Even during purification periods lasting two weeks, many enzymes lose no appreciable activity at 4 C. At the final stage of purification the enzyme preparation is typically dialyzed into a 50% glycerol torage buffer and subsequently stored at -20C. At this temper ature the glycerol solution does not freeze. Most enzymes are stable for well over a 12-month period when properly stored In one stability test of 170 restriction enzymes, activity was assessed after storage for 16 hours at room temperature Of the enzymes tested, 122 (or 72%)exhibited no loss in activity(McMahon, M and Krotee, S, unpublished observation). This point is important to note in case of freezer malfunction Even under optimal storage conditions, however, some enzy may begin to lose noticeable activity within a six-month period The supplier's expiration date, Certificate of Analysis, or catalog will provide more specific information regarding these enzymes. It is best to use these enzymes within a reasonable amount of time after they have been received. Some users employ a freezer box designed to maintain a constant temperature(for short periods at he bench)to store enzymes within the freezer. Alternatively, most enzymes can be stored at C for extended periods. Repeated freeze-thaw cycles from.C to oC is not recommended. Each time the enzyme preparation solution is frozen, the buffer comes out of solution prior to freezing. As a result some enzymes may lose significant activity each time a freeze-thaw cycle is repeated Often the extent of an enzyme's stability during storage at -20C is buffer-related. Identical enzyme preparations obtained from two suppliers, when maintained in their respective storage buffers, may have significantly different shelf lives. How Stable are Diluted Restriction Enzymes? For a discussion, refer above to the question What Can You do to Reduce the Cost of working with Restriction Enzymes SIMPLE DIGESTS How Should You Set up a Simple Restriction Digest: Reaction Condition Most restriction digests are designed either to linearize cloning vector or to generate DNA fragments by cutting a given target DNA to completion at each of the corresponding restric- tion sites To ensure success in any subsequent manipulations (i.e 236 Robinson et al

How Stable Are Restriction Enzymes? As a class, most restriction enzymes are stable proteins. Even during purification periods lasting two weeks, many enzymes lose no appreciable activity at 4°C. At the final stage of purification, the enzyme preparation is typically dialyzed into a 50% glycerol storage buffer and subsequently stored at -20°C. At this temper￾ature the glycerol solution does not freeze. Most enzymes are stable for well over a 12-month period when properly stored. In one stability test of 170 restriction enzymes, activity was assessed after storage for 16 hours at room temperature. Of the enzymes tested, 122 (or 72%) exhibited no loss in activity (McMahon, M., and Krotee, S., unpublished observation). This point is important to note in case of freezer malfunction. Even under optimal storage conditions, however, some enzymes may begin to lose noticeable activity within a six-month period. The supplier’s expiration date, Certificate of Analysis, or catalog will provide more specific information regarding these enzymes. It is best to use these enzymes within a reasonable amount of time after they have been received. Some users employ a freezer box designed to maintain a constant temperature (for short periods at the bench) to store enzymes within the freezer.Alternatively, most enzymes can be stored at -70°C for extended periods. Repeated freeze–thaw cycles from -70°C to 0°C is not recommended. Each time the enzyme preparation solution is frozen, the buffer comes out of solution prior to freezing. As a result some enzymes may lose significant activity each time a freeze–thaw cycle is repeated. Often the extent of an enzyme’s stability during storage at -20°C is buffer-related. Identical enzyme preparations obtained from two suppliers, when maintained in their respective storage buffers, may have significantly different shelf lives. How Stable Are Diluted Restriction Enzymes? For a discussion, refer above to the question What Can You Do to Reduce the Cost of Working with Restriction Enzymes. SIMPLE DIGESTS How Should You Set up a Simple Restriction Digest? Reaction Conditions Most restriction digests are designed either to linearize a cloning vector or to generate DNA fragments by cutting a given target DNA to completion at each of the corresponding restric￾tion sites. To ensure success in any subsequent manipulations (i.e., 236 Robinson et al

ligation), the enzyme treatment must leave each of the resulting DNA termini elements intact To l ug of purified DNA in 50ul of lx reaction buffer, 1 ul of enzyme is added and the reaction is incubated for one hour at the recommended reaction temperature. In most instances the amount of dna can be safely varied from about 250ng to several micrograms and the volume can be varied between 20 ul and 100ul Suitable reaction times may be as little as 15 minutes or as long as 16 hours. Common DNA purification protocols, as well as commercially available kits, yield DNa that is suitable for most digestions. Most commonly used restriction enzymes are of high purity, inexpensive, and provided at concentrations of 5 to 20 units/ul. Using l to 2ul will overcome any expected variability in DNA source, quantity, and purity. The length of incubation time may be decreased to save time or increased to ensure complete digestion of the last few tenths of a percent of substrate, as the reaction asymptotically approaches completion Control reactions Aside from the mere discipline of maintaining"good laboratory practice, the ultimate savings realized in time and effort by running a simple control reaction is often underestimated. Control reactions can often reveal the cause of a failed digest or point to the step within a series of reactions responsible for generating an unexpected result. For every experimental restriction enzyme reaction set performed, a control reaction (containing sample DNA, reaction buffer, and no restriction enzyme) should also be included and analyzed on the agarose gel. Degradation of DnA in the control reaction may indicate nuclease contamination in the DNA preparation or in the buffer. The control reaction products run alongside the sample reaction products on the agarose gel enables for a more accurate assessment of whether the reaction went to completion. Running the appropriate size markers is also recommended Is It Wise to Modify the Suggested Reaction Conditions? Suppliers devote considerable effort in formulating specific enzyme preparations and the corresponding reaction buffers order to ensure sufficient enzyme activity for most common appli cations. In addition suppliers often provide data(Activity Table indicating the relative activity of each enzyme when incubated under standard reaction conditions for a variety of reaction buffers provided. This is a useful guide when planning multiple Restriction Endonucleases 237

ligation), the enzyme treatment must leave each of the resulting DNA termini elements intact. To 1mg of purified DNA in 50ml of 1¥ reaction buffer, 1ml of enzyme is added and the reaction is incubated for one hour at the recommended reaction temperature. In most instances the amount of DNA can be safely varied from about 250ng to several micrograms and the volume can be varied between 20ml and 100ml. Suitable reaction times may be as little as 15 minutes or as long as 16 hours. Common DNA purification protocols, as well as commercially available kits, yield DNA that is suitable for most digestions. Most commonly used restriction enzymes are of high purity, inexpensive, and provided at concentrations of 5 to 20 units/ml. Using 1 to 2ml will overcome any expected variability in DNA source, quantity, and purity.The length of incubation time may be decreased to save time or increased to ensure complete digestion of the last few tenths of a percent of substrate, as the reaction asymptotically approaches completion. Control Reactions Aside from the mere discipline of maintaining “good laboratory practice,” the ultimate savings realized in time and effort by running a simple control reaction is often underestimated. Control reactions can often reveal the cause of a failed digest or point to the step within a series of reactions responsible for generating an unexpected result. For every experimental restriction enzyme reaction set performed, a control reaction (containing sample DNA, reaction buffer, and no restriction enzyme) should also be included and analyzed on the agarose gel. Degradation of DNA in the control reaction may indicate nuclease contamination in the DNA preparation or in the buffer. The control reaction products run alongside the sample reaction products on the agarose gel enables for a more accurate assessment of whether the reaction went to completion. Running the appropriate size markers is also recommended. Is It Wise to Modify the Suggested Reaction Conditions? Suppliers devote considerable effort in formulating specific enzyme preparations and the corresponding reaction buffers in order to ensure sufficient enzyme activity for most common appli￾cations. In addition suppliers often provide data (Activity Table) indicating the relative activity of each enzyme when incubated under standard reaction conditions for a variety of reaction buffers provided. This is a useful guide when planning multiple Restriction Endonucleases 237

restriction enzyme digests. For enzymes with low activity in these standard buffers, specialized buffers are typically supplied Restriction enzymes also have a broad range of activity in nonchloride salt buffers. Some suppliers also offer a potassium acetate or potassium-glutamate single-buffer system that is for mulated to be compatible with a significant subset of their enzymes. (McClelland et al., 1988: O Farrell, Kutter, and Nakanishe, 1980). The reaction buffers themselves are typically supplied as concentrated solutions, ranging from 2x to 10x, and should be properly mixed upon thawing prior to final dilution It is important to note that the reaction buffer supplied with a given enzyme is the same buffer in which all quality assurance assays are performed, and documented in the Certificate of Analy sis provided. Consequently certain modifications to the recom mended reaction conditions(i. e, adding components or changing reaction volume, temperature, or time of incubation) may produce unexpected results. Restriction enzymes can vary considerably in sensitivity to particular changes in their reaction parameters. While salt concentration may have a significant effect on activity, alt type (i.e, NaCl vS KCI)is usually not critical. One exception would be in the case of Smal, which has a strong preference for KCl. For most sensitive enzymes the Certificate of Analysis will detail any reaction modifications not recommended as well as any suggestions for alternative reaction conditions. In order to deter- mine whether a given enzyme may be sensitive to an intended variation in reaction conditions, the Activity Table is also a useful reference. As a rule the most robust enzymes exhibit high relative activity across the range of buffers listed(Pvull). Conversely those enzymes showing a narrow range for high activity may require additional consideration prior to any change in reaction conditions(Sall) and the technical resources provided by the plier should be consulted. All restriction enzymes, as do most other nucleases, requ Mg+ as a cofactor for the dNa cleavage reaction; most buffers for restriction enzymes contain 10mMMg+. To protect DNA preparations in storage buffer from any trace nucleases, EDTA ( Mg+ chelator) is used, often stocked as a disodium salt solution. This is commonly used in various stop-dye solutions as well as electrophoresis buffer. DNA preparations with excessive con- centrations of EDTA may inhibit restriction endonuclease cleav- age, especially if the DNA solution represents a high proportion of the final reaction volume. Addition of Mg+ will alleviate the inhibition Robinson et al

restriction enzyme digests. For enzymes with low activity in these standard buffers, specialized buffers are typically supplied. Restriction enzymes also have a broad range of activity in nonchloride salt buffers. Some suppliers also offer a potassium￾acetate or potassium-glutamate single-buffer system that is for￾mulated to be compatible with a significant subset of their enzymes. (McClelland et al., 1988; O Farrell, Kutter, and Nakanishe, 1980). The reaction buffers themselves are typically supplied as concentrated solutions, ranging from 2¥ to 10¥, and should be properly mixed upon thawing prior to final dilution. It is important to note that the reaction buffer supplied with a given enzyme is the same buffer in which all quality assurance assays are performed, and documented in the Certificate of Analy￾sis provided. Consequently certain modifications to the recom￾mended reaction conditions (i.e., adding components or changing reaction volume, temperature, or time of incubation) may produce unexpected results. Restriction enzymes can vary considerably in sensitivity to particular changes in their reaction parameters. While salt concentration may have a significant effect on activity, salt type (i.e., NaCl vs. KCl) is usually not critical. One exception would be in the case of SmaI, which has a strong preference for KCl. For most sensitive enzymes the Certificate of Analysis will detail any reaction modifications not recommended as well as any suggestions for alternative reaction conditions. In order to deter￾mine whether a given enzyme may be sensitive to an intended variation in reaction conditions, the Activity Table is also a useful reference. As a rule the most robust enzymes exhibit high relative activity across the range of buffers listed (PvuII). Conversely, those enzymes showing a narrow range for high activity may require additional consideration prior to any change in reaction conditions (SalI) and the technical resources provided by the sup￾plier should be consulted. All restriction enzymes, as do most other nucleases, require Mg2+ as a cofactor for the DNA cleavage reaction; most buffers for restriction enzymes contain 10 mM Mg2+ . To protect DNA preparations in storage buffer from any trace nucleases, EDTA (a Mg2+ chelator) is used, often stocked as a disodium salt solution. This is commonly used in various stop-dye solutions as well as electrophoresis buffer. DNA preparations with excessive con￾centrations of EDTA may inhibit restriction endonuclease cleav￾age, especially if the DNA solution represents a high proportion of the final reaction volume. Addition of Mg2+ will alleviate the inhibition. 238 Robinson et al