Plasmid construction by “forced” or “directional” ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCR fragment. To test cleavage efficiency indirectly, a “monitor” plasmid is added to the digest. In a suitable monitor, the two test sites are separated by enough DNA (approximately 20% of full length) to distinguish the double digest from the failed single digest. To make this applicable to combinations of 32 popular cloning enzymes, we constructed a set of 4 monitors (pDM1, pDM2, pDM3, and pDM4). Each contains three polylinkers separated by stuffer segments of approximately 1 kb. The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone. The plasmids also serve as versatile self-monitoring cloning vectors for any site combination.
Most plasmid construction techniques favor the insertion of a DNA fragment over the recircularization of an empty vector (1,2,3,4,5). Examples of these techniques include the use of dephosphorylated vectors (1,2); the use of TA™ and TOPO™ vectors (3,4) for cloning PCR products; ligation of partially end-filled vector restriction sites to other partially end-filled insert sites (e.g., XhoI and BamHI end-filled with TC and GA, respectively); and the forced ligation of vector and insert fragments digested with a pair of enzymes, resulting in directional cloning (5).
Forced ligation is probably the most widely used method of plasmid construction, because of its versatility and efficiency and because it allows for control over the orientation of the insertion. It also results in a high percentage of plasmid clones with inserts (often >90%). However, if one of the two vector insertion sites is cleaved inefficiently, recircularization dominates, and empty vectors are disproportionately represented among transformants. The problem is aggravated when insert concentration is low or when three-fragment ligations are planned.
A successful construction is generally ensured if cleavage of both vector sites can be demonstrated. However, this is not possible if the sites are too close together (e.g., in a vector polylinker) because the linear molecules resulting from a single or double cut are unresolved on a gel. Similarly, digestion of cloning sites incorporated at the end of a PCR product is undetectable. Thus, it is helpful to have an indirect way of determining whether the four sites to be joined in a forced ligation have been cleaved efficiently.
A convenient test for the double digestion of a vector or PCR product is based on the addition of a monitor plasmid to the digest in question. In the monitor, the two sites are located far enough apart to detect both cleavages in the gel pattern of the mixed digest. The applicability of this method has been limited in the past because monitor plasmids suitable for most enzyme pairs were unavailable. Here we describe a set of four monitor plasmids containing 32 commonly used restriction sites, distributed in such a way that double digestion by nearly all combinations can be tested. The same plasmids can also be used as versatile cloning vectors.Materials and Methods Construction of pDM1, pDM2, pDM3, and pDM4
Construction of the pDM plasmids is described at http://www.BioTechniques.com/June2004/AnandSupplementary.html . The pDM plasmids are available on request from the authors.Enzyme digestion, Ligation, and Transformation
Restriction digests of vectors and PCR products were set up in 80 µL volumes, to one half of which was added 0.2 µg of the appropriate pDM monitor plasmid. pDM monitor plasmids were purified by alkaline lysis as previously described, with some modification. (For details of DNA purification, ligation, and transformation, see Supplementary Material.)Results and Discussion Rationale for Monitoring Double Digestions
Successful application of forced ligation depends on efficient double digestion. This is quite difficult to demonstrate directly in a vector polylinker or at the ends of a PCR product. However, it is important to have some way of ensuring double cleavage because there are several potential causes of enzyme failure: DNA preparations may contain inhibitors, buffer conditions may not be optimal for both enzymes (despite vendor assurances), and dated enzymes may be inactive. We routinely test efficiency by including in the digest a monitor plasmid, in which the two cleavage sites are far enough apart to permit the resolution of the doubly and singly digested molecules on an agarose gel. The same technique is used to monitor the digestion of PCR productsConstruction of a Double Digest Monitor Plasmid Set
The four-plasmid pDM monitor set ((Figure 1)) includes 32 commonly used six-base restriction sites, including those in the pUC and pBS polylinkers, plus 15 others. The design criteria for a useful monitor set are (i) that the test sites be unique in each plasmid and (ii) that, for any given pair of restriction enzymes, there is at least one plasmid in the set in which the two sites are well separated. The 4.1-kb pDM construct contains three polylinker segments separated by approximately 1 kb restriction site-free segments, such that cleavage in two of the polylinkers yields approximately 1- and 3-kb fragments or two 2-kb fragments, all well-resolved from the 4.1-kb linear molecule.