Simple Method for Adapting DNA Fragments and PCR Products to All of the Commonly Used Restriction Sites

A problem frequently encountered during the construction of new plasmid vectors is the lack of suitable restriction sites for the insertion of foreign DNA. This problem is particularly common during the cloning of DNA restriction fragments into retroviral and eukaryotic expression vectors, as these vectors are often very large with few cloning sites available. The standard approach is to modify the DNA fragments either by ligation to synthetic oligonucleotide adaptors or linkers, or by creation of blunt-end DNAs using DNA polymerase or exonuclease (3). Both of these approaches can be quite inefficient and expensive. Furthermore, the procedure needs to be repeated if one wishes to adapt the DNA fragment for a different restriction site. Our laboratory has attempted to solve this problem by creating a cloning system (KiSS) that can adapt DNA fragments in a single step to many of the most commonly used restriction sites. This system consists of two plasmids, Kpn-KiSS-lambda and Sac-KiSS-lambda (Figures 1 and 2). Each of these plasmids, derived from pBluescript® II SK(+) and KS(+) (Stratagene, La Jolla, CA, USA), has two sets of multiple cloning sites containing 23 restriction sites. The two sets of multiple cloning sites are positioned as inverted repeats of each other and are separated by a stuffer fragment derived from lambda DNA. DNA inserts, which have compatible overhangs with just one of the 23 restriction sites in the pBluescript SK(+) and KS(+) multiple cloning sites, can be easily adapted for any of the remaining 22 sites by first cloning the insert into one of the KiSS vectors and then cutting it back out using any pair of restriction sites flanking the insert. Figure 3 shows KpnI and SacI fragments, derived from lambda DNA, that have been adapted for four other sites. DNA inserts derived from digestion with two different restriction enzymes may also be adapted using our system. This is accomplished by preparing the appropriate KiSS vector with a complete digestion of the inner sites followed by a partial digestion of the outer sites. The DNA insert can then be ligated to the partially digested vector and excised using any pair of the flanking sites. DNA fragments that cannot be conveniently adapted using the above approach can be adapted by using blunt-end ligations. The DNA fragment can be made blunt-ended by treatment with either DNA polymerase or exonuclease. This is followed by ligation into the blunt-ended EcoRV site of the KiSS vector. The insert can then be cut back out with the restriction enzyme of choice. This is often more efficient than direct blunt-end ligation of the inserts with expression vectors, because the KiSS vectors are small and can simultaneously adapt DNA for multiple sites. The KiSS vectors can also be used for direct cloning of unmodified polymerase chain reaction (PCR) products by converting them into T-vectors (1). This is achieved by incubating EcoRVdigested KiSS vectors with Taq DNA polymerase and deoxythymidine triphosphate (dTTP) (1). The advantages of this approach, compared to using a commercially available TA cloning kit, are that it is inexpensive and that the PCR products are already adapted for all of the commonly used restriction sites, thus making subsequent manipulation of these PCR products easier. The stuffer fragment serves several functions. First, it stabilizes the plasmids by preventing the formation of DNA secondary structures between the inverted, repeated sequences. Such structures could potentially inhibit DNA polymerase function. Second, the