1. Quality Control Assays
2. Activity Assay
3. Overdigest Assay
4. Cut-Ligate-Recut Assay (C-L-R)
5. Blue/White Cloning Assay
6. Genome Qualified
7. Other Assays
8. Quality Control Specifications
9. References

A. Quality Control Assays

During the purification process, restriction enzymes must be separated from a variety of contaminants that can interfere with downstream applications. Even if the restriction enzyme is purified from the native strain or expressed from a clone, the same variety of contaminants are likely to exist and their removal must be demonstrated in the final product. However, performing every possible assay and testing every possible downstream application is potentially redundant and may not provide any useful additional information. With over 20 years of manufacturing and quality control experience, Promega has developed a comprehensive, sensitive and efficient set of contaminant assays and applications tests. Standard assays performed by all restriction enzyme vendors include, activity, overdigest and cut-ligate-recut assays. These are based on interpretation of ethidium bromide-stained DNA bands in agarose gels. Promega uses rigorous criteria in assay interpretation and is dedicated to exceeding, not merely meeting specifications for its enzymes. We strive to ensure that Promega restriction enzymes will surpass customer expectations in every laboratory, every time.

There are several types of contaminants that can be detrimental to both restriction enzyme digests and downstream applications. Both nonspecific and specific (e.g., another restriction enzyme) endonucleases are possible contaminants. These result in undesired cleavage and confusing band patterns on gels. Significant nonspecific exonuclease activity also must be removed. Exonucleases may be active on double-stranded DNA, 5´ single-stranded overhangs or 3´ single-stranded overhangs. The exonuclease activity may exist as part of a polymerase or as a distinct enzyme. At its worst, exonuclease contamination can cause DNA fragments to become completely hydrolyzed or appear as smears on a gel. Even small amounts of exonuclease, able to remove a single base from a restriction fragment, may prevent subsequent ligation and/or recutting of restriction fragments. Phosphatases, another possible contaminant present in all crude cell lysates, will also prevent ligation if present in a restriction digest. Conversely, specific or non-specific nickase activity is rarely seen at any significant level with purified restriction enzymes. Random nicks are much more likely to be caused during DNA preparation and are rarely caused by nickase activity in restriction enzyme preparations.

Star activity deserves special mention. Although this is an inherent activity of the restriction enzyme itself and not a contaminant, the result of star activity is a loss of fidelity and cleavage at undesired and unexpected sequences. This may present the same problems as contamination with a second restriction enzyme. Some vendors do not include information on star activity in their specifications or when reporting quality control (QC) overdigest assay results. Promega's overdigest specification is inclusive of any undesired cutting or exonuclease activity, including star activity. Star activity is best minimized through the avoidance of certain conditions during restriction enzyme purification and by using optimized reaction conditions when performing digests.

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B. Activity Assay

One unit of restriction enzyme activity is defined as the amount of enzyme required to completely digest 1µg of a specified DNA in a total reaction volume of 50µl in 1 hour using the recommended Reaction Buffer and incubation temperature. Results are determined by agarose gel electrophoresis followed by ethidium bromide staining of the restriction fragments. Lambda DNA is the preferred substrate as it is well characterized, inexpensive and readily available at high quality, allowing customers to check unit activity easily. Linear substrates, such as lambda, also tend to give more reproducible results. For enzymes sensitive to dam or dcm methylation, unmethylated lambda that is produced from dam^- or dcm^- E. coli strains is used. If there are no recognition sites (or very few) on lambda, Adenovirus-2 (Ad-2) is usually the next best choice for a unit activity substrate. Promega also uses Ad-2 for some enzymes as it results in a more conservative activity definition. For example, Sal I has three sites in the smaller molecule of Ad-2 and two sites in the larger lambda DNA. There are many more sites in 1µg of Ad-2 than in 1µg of lambda. Therefore, 1 unit of Sal I as defined on Ad-2 constitutes more enzyme than 1 unit as defined on lambda. Lambda predigested with another restriction enzyme may be used to improve the separation of bands seen on the gel when the enzyme of interest has only one or two sites. Although generally avoided, a circular substrate such as pBR322, phiX174, or SV40 may be used for a few enzymes. At Promega, multiple activity assays are performed by more than one scientist. All assays must result in at least the stated label activity. Our re-assay program, where inventory tubes of enzyme are periodically taken from each batch and assayed, guarantees full activity whether the first tube or the last tube of an enzyme batch is purchased.

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C. Overdigest Assay

The overdigest assay is used to assess general enzyme purity. Excess amounts of enzyme are incubated under the same conditions as the activity assay (1µg DNA, 50µl volume, in the same buffer and at the same temperature). In addition to excess enzyme, overdigest assays involve 16-hour incubations. In a few cases, a substrate different from that of the activity assay is used because it is more sensitive to contaminants. Fragments are analyzed on ethidium bromide-stained agarose gels. The greatest number of enzyme units resulting in clear and sharp banding is designated the assay endpoint. The minimum specification varies from enzyme-to-enzyme. Overdigest assays are used to detect unwanted endonuclease, exonuclease and star activity. Enzymes with low overdigest values (<20 units) should be used carefully if an excess amount of enzyme or long incubation times are needed.

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D. Cut-Ligate-Recut Assay (C-L-R)

The ligation assay determines the functional purity of the DNA after restriction enzyme digestion. Substrate DNA is completely digested by incubation with a four-fold excess of enzyme under appropriate assay conditions, ligated with T4 DNA Ligase and recut with the same restriction enzyme. Cut, ligated and recut samples are analyzed by agarose gel electrophoresis followed by ethidium bromide staining. The extent of ligation and recutting are estimated as a qualitative measure of enzyme purity. Specifications are usually ³ 90 or 95% for ligation and ³ 90 or 95% for recutting. This assay demonstrates the integrity of the DNA ends after digestion and an absence of exonuclease (double- or single-stranded) or phosphatases. Although not currently understood, enzymes leaving one base overhangs do not ligate nearly as well as those having blunt ends or ³ 2-base overhangs.

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E. Blue/White Cloning Assay

The Blue/White assay has excellent sensitivity for exonucleases and phosphatases. In addition, it is a quantitative measure that provides verification of cloning performance. This assay is performed on enzymes that cut at a single site in the multiple cloning region of one of the pGEM^® Vectors^(a). The multiple cloning sequence of these vectors is embedded within a coding sequence for the lacZ gene alpha-peptide. The pGEM^® Vectors also contain a selectable marker such as ampicillin resistance. During the blue/white cloning assay the plasmid is several-fold overdigested with the enzyme, the DNA is ligated (without insert) and then transformed into cells lacking the lacZ alpha-peptide. An agarose gel of cut, ligated and recut DNA is also examined. If the integrity of the cut ends is perfectly maintained, ligation will produce mostly higher molecular weight concatamers and a lesser amount of circularized monomer. Upon transformation and alpha-peptide expression, the functional lacZ gene product (beta-galactosidase) is produced through alpha-complementation. When the transformants are plated on IPTG and X-Gal, blue colonies result. Expected transformation efficiency is generally 1-2 orders of magnitude lower than with uncut control plasmid. Both phosphatase and exonuclease contamination will lower ligation efficiency and thereby decrease transformation efficiency. More importantly, loss of nucleotides at the cut site, even if ligatable, yields a mixed population of clones containing frame shifts and codon deletions in the lacZ alpha-peptide. These result in nonfunctional alpha-peptides and generate white colonies, potential false positives in a cloning experiment (1). A special case is the loss of a single nucleotide. In this instance the resultant colonies are able to use an alternative start codon that shifts to become in-frame. However, this produces weak translation initiation and/or improper complementation for fully active beta-galactosidase and the colonies develop a faint blue color, which is easily mistaken for white. An as yet unidentified contaminant that removes the 3´ nucleotide from cut DNA and causes faint or pale blue colonies exists even in restriction enzymes prepared from clones. This is especially problematic with blunt end cutting enzymes. Not all commercial vendors specifically assay for and remove this contaminant (2). The Promega specification for enzymes producing overhangs is <2% white or pale blue colonies compared with undigested plasmid controls and <5% white or pale blue colonies for enzymes producing blunt ends.

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F. Genome Qualified

A number of Promega’s restriction enzymes are useful in chromosomal mapping due to the low frequency of their recognition sites. Promega's genome qualified restriction enzymes are assayed to ensure optimal performance in genome analysis applications. These enzymes have been tested on chromosomal DNA templates embedded in agarose plugs. The resultant digests must yield clear and complete digestion patterns in pulsed field gels stained with ethidium bromide. See Digestion of High Molecular Weight DNA for further information on this topic.

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G. Other Assays

Promega uses a number of assays that may not be part of the final QC specifications. Exonuclease assays using tritiated substrates are sometimes used during purification because of their speed and ease-of-use. However, these do not approach the single nucleotide sensitivity of the blue/white assay. Also, small digestion products (<30bp) may not precipitate efficiently and lead to erroneously high release values. Small oligonucleotides end-labeled with ³²P may be used for detecting phosphatase activity or removal of single nucleotides, but fidelity of restriction fragment ends is more practically demonstrated by the cut-ligate-recut and blue/white assays. Assays for topoisomerases and nickases may also be used during purification but these potential contaminants are easily eliminated before the final product is prepared.

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H. Quality Control Specifications

The quality control acceptance criteria for Promega's restriction enzymes are listed in Table 1.6. Routinely, the actual values surpass the quality standards listed.

Table 1.6. Restriction Enzyme Quality Control Specifications.

Aat to Bgl | Bsa to Csp | Dde to I-Ppo | Kpn to Rsa | Sac to Xmn

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I. References

1. Hung, L. et al. (1991) A blue/white cloning assay for quality control of DNA restriction and modifying enzymes. Promega Notes 33, 12.
2. Murray, E. et al. (1993) Cloning-qualified blunt end restriction enzymes: Causes and cures for light blue colonies. Promega Notes 41, 1.

pGEM is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office.