High-efficiency cloning system for versatile adaptation of DNA fragments.

Recombinant DNA vectors are fundamental tools in molecular biology and genetic engineering. One of the problems frequently encountered in vector construction is the lack of compatible restriction sites between the vectors and the DNA inserts. The conventional approach to solve this problem is to make the DNA fragments blunt-ended (3,4,12) or to add linkers or adaptors (10,11,14) before ligation. However, the efficiency of using blunt-end ligation and linkers/adaptors is low. The addition of linkers or adaptors can be expensive because a new linker/ adaptor has to be made for insertion at each new restriction site. Moreover, they are not commercially available for all sites. As PCR is one of the most important techniques in the field of recombinant DNA, the introduction of new restriction sites to DNA fragments by PCR has become an alternative method (1,6,13). However, PCR-directed mutagenesis requires prior knowledge of the flanking sequence of the targeted fragments to design the primers and to synthesize the oligonucleotide. It also takes time to characterize the optimal conditions before large-scale PCR amplification. The main disadvantages of PCR-generated mutagenesis are as follows. (i) There exists a size limitation on the fragments that can be amplified by PCR. Introduction of new restriction sites by PCR may not be applicable to large DNA fragments. The size limitation of DNA fragments obtained by high-fidelity PCR fragments is less than 5 kb (2). Although currently longer and more accurate DNA amplification can be achieved by using high-fidelity DNA polymerases with 3′→5′ proofreading exonuclease activity (2,8), amplifying fragments in excess of 20 kb in length is still problematic, especially from complex templates such as genomic DNA (1,2). (ii) Sequence analysis is required for each PCR-amplified fragment (1). Even with high-fidelity DNA polymerases, the entire amplified fragment has to be sequenced to insure there are no DNA polymerase-derived mutations. Furthermore, several rounds of sequence analysis are required for any fragment larger than 800 bp because the reliable sequence obtained from each sequencing reaction is 500–800 bp (1). New primers have to be synthesized for each round of sequencing analysis. (iii) Primer design and synthesis, as well as the entire PCR process, have to be repeated if the same DNA fragment needs to be inserted into different sites. To overcome these difficulties, our laboratory has developed a novel cloning system, pLinus. By using simple, inexpensive, and efficient basic cloning techniques, pLinus vectors can adapt DNA fragments from various sources in a single step to 32 unique restriction sites simultaneously, thus making the repeat procedure unnecessary. Here we discuss the features and advantages of this user-friendly pLinus cloning system. The pLinus system consists of two sets of plasmids, pLinus16 (Figure 1, A and B) and pLinus 17 (Figure 2, A and B). Both pLinus16 and pLinus17 consist of a pair of plasmids derived from pLitmus 28/29 and 38/39 (New England Biolabs, Beverly, MA, USA) respectively. They provide a total of 29 unique restriction sites with cohesive ends and three restriction sites (SnaBI, EcoRV, and StuI) for blunt-end ligations. Each pLinus plasmid contains two inverted repeated copies of the multiple cloning sites flanking a central stuffer fragment derived from either λ DNA or enhanced GFP (EGFP) sequence. The 780-bp stuffer piece in pLinus 16-Apa was an EGFP gene fragment, cut with restriction endonuclease ApaI from the pcDNA3.1-EGFP plasmid (data not shown). The pcDNA3.1-EGFP was constructed by insertion of a 758-bp KpnI/ NotI EGFP fragment from pEGFP-1 (BD Biosciences Clontech, Palo Alto, CA, USA) into the KpnI/NotI sites of pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). The two inverted repeated copies of the multiple cloning site in each pLinus vector allow versatile insertion of DNA fragments and provide a cassette such that DNA pieces can be cut out and cloned into other vectors, thus making the subsequent handling of DNA pieces