( Reliable res...)Restriction Endonucleases Undergo Rigorous QC Testing to Ensure Optimal,,,Performance

Reliable restriction enzymes satisfy all your research needs

Ronda Allen Kim Kuplent
Stratagene

Restriction endonucleases are used in many molecular biology protocols, making the functional purity of critical importance. Each restriction enzyme preparation must be free of contaminants that can potentially interfere with either DNA cleavage or subsequent reactions that use digested DNA (i.e., ligation or sequencing reactions). Such contaminants might include other site-specific endonucleases that produce unwanted DNA fragments, exonucleases that destroy the integrity of the cut site by cleaving nucleotides from the resultant 5 or 3 termini of the fragments, and phosphatases that inhibit ligation reactions by removing the 5 phosphate from DNA ends. To ensure that Stratagenes enzymes consistently meet the needs of the research community, we impose strict quality control procedures in analyzing every enzyme preparation. In most instances, the testing conditions are designed to be more rigorous than typical laboratory applications. In this article, we describe the assays used to establish the functional purity of restriction endonuclease preparations.

Unit Activity Determination

Prior to assaying samples for contaminants, the unit activity of each enzyme sample is accurately determined. One unit of activity is defined as the amount of enzyme required to completely digest 1 g of substrate DNA in 1 hour at the appropriate temperature under the optimal assay conditions. This information is provided on the Certificate of Analysis provided with each restriction endonuclease. Restriction enzyme activity is substrate-dependent; therefore, when digesting a new substrate, we recommend titrating the amount of enzyme added. The unit activity of a representative sample from each packaging of a restriction endonuclease is confirmed prior to shipment to demonstrate that no loss of enzymatic activity occurs upon packaging.

Overdigestion of Substrates

When enzymes are tested under standard digestion conditions (1 U of enzyme/g substrate DNA), trace levels of contaminating DNase activities might not be detected. To more stringently assess the functional purity of each enzyme preparation, Stratagene incubates a 4- to 100-fold excess of enzyme with substrate DNA. After a 16-hour incubation at the appropriate temperature, the banding pattern from these overdigested samples is compared to the signature-banding pattern produced by the enzyme in 1 hour. If the restriction endonuclease preparation is free from contaminating endonuclease and exonuclease activities, the overdigested reactions will exhibit the identical banding pattern characteristic of the 1-hour reactions. The maximum number of units that clearly exhibit this pattern in the overdigestion reactions is reported on the Certificate of Analysis provided with each enzyme. This overdigestion assay is also employed on a representative sample following aliquoting and packaging to confirm that contaminating DNase activity is not introduced during packaging.

Detecting Exonuclease Contaminants

The integrity of a restriction enzyme cut site can be destroyed by the presence of exonucleases that can digest single-or double-stranded ends. Stratagene specifically assays for exonuclease activity by incubating an excess of restriction endonuclease for 4 hours with a mixture of single- and double-stranded, [³H]-thymidine-labeled genomic E. coli DNA. Contaminating exonuclease is indicated if over 0.6% of the ³H-labeled DNA added to the reactions is detected as trichloroacetic acid-soluble counts.

Ligation Assay

Figure 1

Stratagene confirms the functional purity of each restriction enzyme preparation using an assay that simulates a typical cloning protocol. In this assay, substrate DNA is completely digested using a three-fold excess of enzyme, and the enzyme is removed using StrataClean resin. The digestion products are ligated using Stratagenes T4 DNA ligase and then recut with the same restriction enzyme. Samples of the cut, ligated, and recut DNA are subsequently analyzed by agarose gel electrophoresis (Figure 1). If the ends of the digested DNA have been compromised by contaminating exonucleases or phosphatases, the recognition site will no longer be intact, and the cut product will not ligate efficiently or, when recut, yield a banding pattern identical to the initial digest. Stratagene reports the percent of ligated and recut substrates on the Certificate of Analysis.

Nicking Assay

Figure 2

To analyze for contaminating endonuclease or topoisomerase activities, Stratagene incubates an excess of restriction endonuclease with supercoiled plasmid DNA that lacks the restriction enzyme recognition sequence. Following a 4-hour incubation at the appropriate temperature, the conversion of supercoiled DNA (RF I) to the open circular form (RF II) is monitored by agarose gel electrophoresis (Figure 2). This conversion is indicative of a single nick in the RF I DNA. If greater levels of contamination are present, linear DNA (RF III) might also be observed. Stratagene guarantees that its restriction endonuclease preparations will generate <5% conversion to RF II DNA and no detectable conversion to RF III DNA.

Blue-White Cloning Assay

Figure 3

Another highly sensitive assay that models the use of restriction enzymes is the blue-white color selection assay. In this assay, Stratagenes pBluescript II SK phagemid vector (Figure 3), which contains the lacZ reporter gene, is cut at a single position within the multiple cloning site using a five-fold excess of enzyme. A phenol-chloroform extraction is used to purify the linearized vector. The vector is ligated and transformed into Epicurian Coli XL1-Blue competent cells, which are then plated on a medium containing X-gal and IPTG. Functional b-galactosidase (determined by blue colonies) is produced only for those transformations with plasmids containing uninterrupted reading frames. If the integrity of the DNA ends produced during the digestion reaction is compromised (i.e., a nucleotide is deleted), the lacZ gene is interrupted, and white colonies result. Enzymes tested in this assay (those commonly used in cloning applications) must produce less than 5% white colonies.

Stability Testing

The unit activity of each restriction endonuclease is confirmed at least once every six months. This stability testing guarantees that enzymes are fully active when delivered to your laboratory. Most Stratagene restriction enzymes are guaranteed to be stable for 18 months from the date of testing.

Conclusions

Stratagene recognizes that high-quality reagents are imperative for molecular biology applications and, therefore, commits to providing an extensive line of rigorously qualified restriction enzymes. We stringently establish the functional purity of every enzyme preparation to ensure optimal performance.