Hsu and Berg, 1978). Prolonged incubation time and high enzyme concentration as well as elevated levels of glycerol and other organic solvents tend to generate star activity (Malyguine Vannier, and Yot, 1980). Maintaining the glycerol concentration to 5% or less is recommended. Since the enzyme is supplied in 50% glycerol, the enzyme added to a reaction should be no more than 10% of the final reaction volume When extra DNA fragments are observed, especially when working with an enzyme for the first time, star activity must be differentiated from partial digestion or contaminating specific endonucleases. first, check to make sure that the reaction condi- tions are well within the optimal range for the enzyme. Then, repeat the digest in parallel reactions, one with twice the activity and one with half the activity of the initial digest. Partial digestion is indicated as the cause when the number of bands is reduced to that expected after repeating the digestion with additional enzyme (or extending incubation time). If extra bands are still evident, contact the supplier's technical support resource for advice. Generally speaking, star activity and contaminating activ ities are more difficult to differentiate Mapping and sequencing he respective cleave es is the best method to distinguish star activity from a partial digest or contaminant activity Site Preference The rate of cleavage at each site within a given DNA substrate can vary(Thomas and Davis, 1975). Fragments containing a subset of sites that are cleaved more slowly than others can result in partial digests containing lighter bands visualized on an ethidium tained agarose gel. Certain enzymes such as Eco Rll require an activator site to allow cleavage(Kruger et al., 1988 ). Substrates lacking the additional site will be cleaved very slowly. For certain enzymes(Nael), adding oligonucleotides containing the site or adding another substrate containing multiple sites can improve utting. In the case of PaeR7l, it has been shown that the sur- rounding sequence can have a profound effect on the cleavage rate(Gingeras and Brooks, 1983). In most cases this rate differ ence is taken in to account because the unit is defined at a point of complete digestion on a standard substrate DNA(e.g, lambda DNA)that contains multiple sites. Problems can arise when certain sites are far more resistant than others, or when highly resistant sites are encountered on substrates other than the stan dard substrate DNA. If a highly resistant site is present in a common cloning vector, then a warning should be noted on the data card or in the catalog Robinson et al
(Hsu and Berg, 1978). Prolonged incubation time and high enzyme concentration as well as elevated levels of glycerol and other organic solvents tend to generate star activity (Malyguine, Vannier, and Yot, 1980). Maintaining the glycerol concentration to 5% or less is recommended. Since the enzyme is supplied in 50% glycerol, the enzyme added to a reaction should be no more than 10% of the final reaction volume. When extra DNA fragments are observed, especially when working with an enzyme for the first time, star activity must be differentiated from partial digestion or contaminating specific endonucleases. First, check to make sure that the reaction conditions are well within the optimal range for the enzyme. Then, repeat the digest in parallel reactions, one with twice the activity and one with half the activity of the initial digest. Partial digestion is indicated as the cause when the number of bands is reduced to that expected after repeating the digestion with additional enzyme (or extending incubation time). If extra bands are still evident, contact the supplier’s technical support resource for advice. Generally speaking, star activity and contaminating activities are more difficult to differentiate. Mapping and sequencing the respective cleavage sites is the best method to distinguish star activity from a partial digest or contaminant activity. Site Preference The rate of cleavage at each site within a given DNA substrate can vary (Thomas and Davis, 1975). Fragments containing a subset of sites that are cleaved more slowly than others can result in partial digests containing lighter bands visualized on an ethidium stained agarose gel. Certain enzymes such as EcoRII require an activator site to allow cleavage (Kruger et al., 1988). Substrates lacking the additional site will be cleaved very slowly. For certain enzymes (NaeI), adding oligonucleotides containing the site or adding another substrate containing multiple sites can improve cutting. In the case of PaeR7I, it has been shown that the surrounding sequence can have a profound effect on the cleavage rate (Gingeras and Brooks, 1983). In most cases this rate difference is taken in to account because the unit is defined at a point of complete digestion on a standard substrate DNA (e.g., lambda DNA) that contains multiple sites. Problems can arise when certain sites are far more resistant than others, or when highly resistant sites are encountered on substrates other than the standard substrate DNA. If a highly resistant site is present in a common cloning vector, then a warning should be noted on the data card or in the catalog. 230 Robinson et al
Methylation Methylation sensitivity can interfere with digestion and cloning eps. Many of the E coli cloning strains express the genes for EcoKI methylase, dam methylase, or dcm methylase. The dam methylase recognizes gatC and methylates at the N6 position of adenine. Mbol recognizes gatC (the same four base-pair sequence as dam methylase) and will only cleave dNa purifie from E coli strains lacking the dam methylase DpnI is one of on a few enzymes known to cleave methylated dNA preferentially, and it will only cleave dNa from dam* strains ( lacks and greer erg, 1977). Another E coli methylase, termed dcm, was found to block AatI and Stul(Song, Rueter, and Geiger, 1988). The dcm methylase recognizes CC(A/T)GG and methylates the second C The restriction enzyme recognition site doesn't have to span the entire methylation site to be blocked. Overlapping methylation sites can cause a problem. An example is the Xbal recognition site 5 TCTAGA 3. Although it lacks the gatC dam methylase or the following 3 bases are TC giving TCTAGATC, then the dam methylase blocks Xbal from cutting. E coli strains with deleted dam and dcm, like GM2163, are commercially available and should be used if the restriction site of interest is blocked by methylation. The first time a methylated plasmid is transformed into GM2163 the number of colonies will be low due to the impor tant role played by dam during replication Methylation problems can also arise when working with mam- malian or plant DNA. DNA from mammalian sources contain C5 methylation at CG sequences. Plant DNA often contains C5 methylation at CG and CNG sequences. Bacterial species contain a wide range of methylation contributed by their restriction mod ification systems(Nelson, Raschke, and McClelland, 1993). Infor mation regarding known sensitivities to methylation can be found on data cards in catalog tables, by searching REBASE, and in the preceding review by Nelson Cloning problems can arise when working with DNA methy lated at the c5 position Most e coli strains have an mcr restric- tion system that cleaves methylated DNA(Raleigh et al., 1988) a strain deficient in this system must be used when cloning DNA from mammalian and plant sources. Substrate effec More on this discussion appears in the question below, How Can a Substrate Affect the Restriction Digest? Restriction Endonucleases 23
Methylation Methylation sensitivity can interfere with digestion and cloning steps. Many of the E. coli cloning strains express the genes for EcoKI methylase, dam methylase, or dcm methylase. The dam methylase recognizes GATC and methylates at the N6 position of adenine. MboI recognizes GATC (the same four base-pair sequence as dam methylase) and will only cleave DNA purified from E. coli strains lacking the dam methylase. DpnI is one of only a few enzymes known to cleave methylated DNA preferentially, and it will only cleave DNA from dam+ strains (Lacks and Greenberg, 1977). Another E. coli methylase, termed dcm, was found to block AatI and StuI (Song, Rueter, and Geiger, 1988). The dcm methylase recognizes CC(A/T)GG and methylates the second C at the C5 position. The restriction enzyme recognition site doesn’t have to span the entire methylation site to be blocked. Overlapping methylation sites can cause a problem.An example is the XbaI recognition site 5¢ TCTAGA 3¢. Although it lacks the GATC dam methylase target, if the preceding 5¢ two bases are GA giving GATCTAGA or the following 3¢ bases are TC giving TCTAGATC, then the dam methylase blocks XbaI from cutting. E. coli strains with deleted dam and dcm, like GM2163, are commercially available and should be used if the restriction site of interest is blocked by methylation. The first time a methylated plasmid is transformed into GM2163 the number of colonies will be low due to the important role played by dam during replication. Methylation problems can also arise when working with mammalian or plant DNA. DNA from mammalian sources contain C5 methylation at CG sequences. Plant DNA often contains C5 methylation at CG and CNG sequences. Bacterial species contain a wide range of methylation contributed by their restriction modification systems (Nelson, Raschke, and McClelland, 1993). Information regarding known sensitivities to methylation can be found on data cards in catalog tables, by searching REBASE, and in the preceding review by Nelson. Cloning problems can arise when working with DNA methylated at the C5 position. Most E. coli strains have an mcr restriction system that cleaves methylated DNA (Raleigh et al., 1988). A strain deficient in this system must be used when cloning DNA from mammalian and plant sources. Substrate Effects More on this discussion appears in the question below, How Can a Substrate Affect the Restriction Digest? Restriction Endonucleases 231
WHAT ARE THE GENERAL PROPERTIES OF RESTRICTION ENDONUCLEASES? In general, commercial preparations of restriction endonucle- ases are purified and stored under conditions that ensure optimal nd stability over time; namely -20oC. They are com monly supplied in a solution containing 50% glycerol, Tris buffer, EDTA, salt, and reducing agent. This solution will conveniently remain in liquid form at -20C but will freeze at temperatures below -30%C. Those enzymes shipped on dry ice, or stored at -70oC, will have a white crystalline appearance; they revert to a clear solution as the temperature approaches -20C. As a rule repeated freeze-thaw cycles are not recommended for enzyme solutions because of the possible adverse effects of shearing(more on the question, How Stable are Restriction Enzymes? appears As a group(and by definition), Class II restriction endonucle ases require magnesium(Mg)as a cofactor in order to cleave DNA at their respective recognition sites. Most restriction enzymes are incubated at 37C, but many require higher or lower (i. e, Smal requires incubation at 25C)temperatures. Percent activity tables of thermophilic enzymes incubated at 37 C can be found in some suppliers'catalogs. For most reactions, the pl optima is between 7 and 8 and the NaCl concentration betwee 50 and 100 mM. Concentrated reaction buffers for each enzyme are provided by suppliers. Typically each enzyme is profiled for optimal activity as a function of reaction temperature, pH(buffer ing systems), and salt concentration. Some enzymes are also evaluated in reactions containing additional components(BSA detergents). Generally, these characteristics are documented in the published literature and referenced by suppliers Interestingly, a number of commonly used enzymes can dis broad range of stability and performance characteristics under fairly common reaction conditions. They may vary considerably i activity and may exhibit sensitivity to particular components. In effort to minimize these undesirable effects, suppliers often adjust enzyme buffer components and concentrations to ensure optimal performance for the most common applications. There is a wealth of information about the properties of these enzymes in most suppliers'catalogs, as well as on their Web sites. The documentation supplied with the restriction endonuclease should contain detailed information about the enzymes pro- perties and functional purity. It is important to read the Certifi cate of Analysis when using a restriction enzyme for the first Robinson et al
WHAT ARE THE GENERAL PROPERTIES OF RESTRICTION ENDONUCLEASES? In general, commercial preparations of restriction endonucleases are purified and stored under conditions that ensure optimal reactivity and stability over time; namely -20°C. They are commonly supplied in a solution containing 50% glycerol, Tris buffer, EDTA, salt, and reducing agent. This solution will conveniently remain in liquid form at -20°C but will freeze at temperatures below -30°C. Those enzymes shipped on dry ice, or stored at -70°C, will have a white crystalline appearance; they revert to a clear solution as the temperature approaches -20°C. As a rule repeated freeze-thaw cycles are not recommended for enzyme solutions because of the possible adverse effects of shearing (more on the question, How Stable are Restriction Enzymes? appears below). As a group (and by definition), Class II restriction endonucleases require magnesium (Mg2+ ) as a cofactor in order to cleave DNA at their respective recognition sites. Most restriction enzymes are incubated at 37°C, but many require higher or lower (i.e., SmaI requires incubation at 25°C) temperatures. Percent activity tables of thermophilic enzymes incubated at 37°C can be found in some suppliers’ catalogs. For most reactions, the pH optima is between 7 and 8 and the NaCl concentration between 50 and 100 mM. Concentrated reaction buffers for each enzyme are provided by suppliers. Typically each enzyme is profiled for optimal activity as a function of reaction temperature, pH (buffering systems), and salt concentration. Some enzymes are also evaluated in reactions containing additional components (BSA, detergents). Generally, these characteristics are documented in the published literature and referenced by suppliers. Interestingly, a number of commonly used enzymes can display a broad range of stability and performance characteristics under fairly common reaction conditions. They may vary considerably in activity and may exhibit sensitivity to particular components. In an effort to minimize these undesirable effects, suppliers often adjust enzyme buffer components and concentrations to ensure optimal performance for the most common applications. There is a wealth of information about the properties of these enzymes in most suppliers’ catalogs, as well as on their Web sites. The documentation supplied with the restriction endonuclease should contain detailed information about the enzyme’s properties and functional purity. It is important to read the Certifi- cate of Analysis when using a restriction enzyme for the first 232 Robinson et al
time, as it may provide important information concerning partic ular substrate DNAs or alternative reaction conditions for a spe cific application What Insight Is Provided by a Restriction Enzyme's Quality Control Data? Restriction enzymes are isolated from bacterial strains that contain a variety of other enzyme activities required for normal cell function. These additional activities include other nucleases phosphatases, and polymerases as well as other dna binding pro- teins that may inhibit restriction enzyme activity In preparations where trace amounts of these activities remain the end-structure of the resulting DNA fragments may be degraded, thus inhibiting subsequent ligation. Likewise plasmid substrates may be nicked thus reducing transformation efficiencies Ideally the restriction enzyme preparation should be purified to homogeneity and free of any detectable activities that might inter fere with digestion or inhibit subsequent reactions planned for the resulting DNA fragments. In order to provide researchers with a practical means to conveniently evaluate the suitability of a given restriction enzyme preparation, suppliers include a Certificate of Analysis with each product, detailing the preparation,s per formance in a defined set of Quality Control Assays. In order to establish a standard reference for the amount of enzyme and sub- strate used in these assays, each supplier must first define the unit substrate and reaction conditions for each product Unit Definition a unit of restriction endonuclease is defined as the amount of zyme required to completely cleave l ug of substrate DNA suS- pended in 50 ul of the recommended reaction buffer in one hour at the recommended assay buffer and temperature. The DNA most often used is bacteriophage Lambda or another well characterized substrate. Note that the unit definition is not based on classic enzyme kinetics. The enzyme molar concentration is in excess. A complete digest is determined by the visualized pattern of cleaved DNA fragments resolved by electrophoresis on an ethidium bromide-stained gel. Some restriction enzymes will behave differently when used outside the parameters of the unit definition. The number of sites(site density)or the particular type of DNA substrate may have an effect on"unit activity, " but it is not always proportional(Fuchs and Blakesley, 1983) Restriction Endonucleases 233
time, as it may provide important information concerning particular substrate DNAs or alternative reaction conditions for a specific application. What Insight Is Provided by a Restriction Enzyme’s Quality Control Data? Restriction enzymes are isolated from bacterial strains that contain a variety of other enzyme activities required for normal cell function. These additional activities include other nucleases, phosphatases, and polymerases as well as other DNA binding proteins that may inhibit restriction enzyme activity. In preparations where trace amounts of these activities remain, the end-structure of the resulting DNA fragments may be degraded, thus inhibiting subsequent ligation. Likewise plasmid substrates may be nicked, thus reducing transformation efficiencies. Ideally the restriction enzyme preparation should be purified to homogeneity and free of any detectable activities that might interfere with digestion or inhibit subsequent reactions planned for the resulting DNA fragments. In order to provide researchers with a practical means to conveniently evaluate the suitability of a given restriction enzyme preparation, suppliers include a Certificate of Analysis with each product, detailing the preparation’s performance in a defined set of Quality Control Assays. In order to establish a standard reference for the amount of enzyme and substrate used in these assays, each supplier must first define the unit substrate and reaction conditions for each product. Unit Definition A unit of restriction endonuclease is defined as the amount of enzyme required to completely cleave 1mg of substrate DNA suspended in 50ml of the recommended reaction buffer in one hour at the recommended assay buffer and temperature. The DNA most often used is bacteriophage Lambda or another wellcharacterized substrate. Note that the unit definition is not based on classic enzyme kinetics. The enzyme molar concentration is in excess. A complete digest is determined by the visualized pattern of cleaved DNA fragments resolved by electrophoresis on an ethidium bromide-stained gel. Some restriction enzymes will behave differently when used outside the parameters of the unit definition. The number of sites (site density) or the particular type of DNA substrate may have an effect on “unit activity,” but it is not always proportional (Fuchs and Blakesley, 1983). Restriction Endonucleases 233
Quality Control Assays-Maximum Units per Reaction When using procedures requiring larger quantities of enzyme and/or extended reaction times, an appreciation of the quality control data can help determine a safe amount of enzyme for your Overnight Assay Increasing amounts of restriction endonuclease are incubated overnight(typically for 16 hours)in their recommended buffer with l ug of substrate dNa in a volume of 50 ul. The characteris- tic limit digest banding pattern produced by the enzyme in one hour is compared to the pattern produced from an excess of enzyme incubated overnight. A sharp, unaltered pattern under these conditions is an indication that the enzyme preparation is free of detectable levels of nonspecific endonucleases. The maximum number of units yielding an unaltered pattern is eported. Enzymes listing 100 units or more, a 1600-fold over digestion(100 units x 16h), will not degrade DNA up to megabase size in mapping experiments and can be assumed to be virtually free of nonspecific endonuclease (Davis, T. and Robinson, D unpublished observations Nicking Assay Another sensitive test for contaminating endonucleases is a four hour incubation with a supercoiled plasmid that lacks a site for the enzyme being tested. The supercoil is very sensi- tive to nonspecific nicking by a single-stranded endonuclease, cleavage by a double-stranded endonuclease, or topoisomerase activity. If a single-stranded nick occurs, the supercoiled mole- cule. RFl. unwinds and assumes the circular form rFil. If a double-stranded cleavage occurs, the circle will become linear High levels of single-stranded nicking leads to linear DNA All three forms of DNa have distinct electrophoretic mobilities on agarose gels. Enzymes converting 5%or less of the plasmid o relaxed form using 100 units of enzyme for four hours can be considered virtually free of nicking activity. High-salt buffers, especially at elevated temperature, can cause some conversion to relaxed form. A control reaction, including buffer and dnA but lacking enzyme, is incubated and run on the agarose gel fo Exonuclease Assay Suppliers use a variety of assays to check for exonuclease activ ity. a general assay mixture contains a restriction endonuclease 34 Robinson et al
Quality Control Assays—Maximum Units per Reaction When using procedures requiring larger quantities of enzyme and/or extended reaction times, an appreciation of the quality control data can help determine a safe amount of enzyme for your application. Overnight Assay Increasing amounts of restriction endonuclease are incubated overnight (typically for 16 hours) in their recommended buffer with 1mg of substrate DNA in a volume of 50ml. The characteristic limit digest banding pattern produced by the enzyme in one hour is compared to the pattern produced from an excess of enzyme incubated overnight. A sharp, unaltered pattern under these conditions is an indication that the enzyme preparation is free of detectable levels of nonspecific endonucleases. The maximum number of units yielding an unaltered pattern is reported. Enzymes listing 100 units or more, a 1600-fold over digestion (100 units ¥ 16h), will not degrade DNA up to megabase size in mapping experiments and can be assumed to be virtually free of nonspecific endonuclease (Davis, T. and Robinson, D., unpublished observations). Nicking Assay Another sensitive test for contaminating endonucleases is a four hour incubation with a supercoiled plasmid that lacks a site for the enzyme being tested. The supercoil is very sensitive to nonspecific nicking by a single-stranded endonuclease, cleavage by a double-stranded endonuclease, or topoisomerase activity. If a single-stranded nick occurs, the supercoiled molecule, RFI, unwinds and assumes the circular form, RFII. If a double-stranded cleavage occurs, the circle will become linear. High levels of single-stranded nicking leads to linear DNA. All three forms of DNA have distinct electrophoretic mobilities on agarose gels. Enzymes converting 5% or less of the plasmid to relaxed form using 100 units of enzyme for four hours can be considered virtually free of nicking activity. High-salt buffers, especially at elevated temperature, can cause some conversion to relaxed form. A control reaction, including buffer and DNA but lacking enzyme, is incubated and run on the agarose gel for comparison. Exonuclease Assay Suppliers use a variety of assays to check for exonuclease activity. A general assay mixture contains a restriction endonuclease 234 Robinson et al