pPCV, a versatile vector for cloning PCR products

The in vitro amplification of DNA fragments by polymerase chain reaction (PCR) is a routine technique in most molecular biology laboratories. Direct cloning of DNA fragments amplified by Taq DNA polymerase has frequently been found to be inefficient [Harrison et al. 1994] since this enzyme tends to add a non-templated nucleotide to the 3′ ends of the amplicon, mostly an adenosine residue, leaving a 3′overhang [Clark ] since this enzyme tends to add a non-templated nucleotide to the 3′ ends of the amplicon, mostly an adenosine residue, leaving a 3′overhang [Clark ] since this enzyme tends to add a non-templated nucleotide to the 3′ ends of the amplicon, mostly an adenosine residue, leaving a 3′overhang [Clark 1988. To circumvent this limitation, some commercially available vectors were constructed in order to have a 3′-T overhang (T-vectors) for sticky-end cloning. Many strategies have been developed to add a 3′-T overhang. One approach involves tailing a blunt-ended vector using terminal transferase in the presence of dideoxythymidine triphosphate (ddTTP) [Holton & Graham 1991 but there is a high probability that some vector molecules will lack an overhang at one or both ends. These incomplete plasmids can circularize during ligation rendering ineffective for cloning [Jun et al. 2010. Another approach is to digest a parental vector with a restriction enzyme that will generate single 3′-T overhangs. Restriction enzymes used for that purpose include Bci VI, Bfi I, Hph I, Mnl I, Taa I, Xcm I and Eam 1105I [Jun et al. 2010; Dimov 2012; Gu & Ye 2011; Borovkov & Rivkin 1997. However, these vectors are not recommended for cloning amplicons produced by DNA polymerases which generate blunt-ended products.

The aim of this work was to construct a vector based on pBlueScript® II KS with a modified polylinker which would allow direct cloning of PCR products bearing either blunt-ends or A-overhangs.

For vector construction, a minimal polylinker was designed (Figure 1A) with the inclusion of restriction sites for Xcm I, which produce 3′-T overhangs that can be used for cloning PCR products derived from amplification by Taq polymerase, and Eco RV, which yields blunt-ends suitable for cloning PCR products generated by Pfu DNA polymerases. It is argued that the use of Xcm I is limited because vectors incubated with this enzyme are often partially digested leading to a high background of non-recombinant transformants [Xuejun et al. 2002]. This issue was solved by the insertion of a stuffer DNA sequence large enough to be easily separated by gel electrophoresis [Gu & Ye 2011; Jo & Jo 2001. The new polylinker still allows blue/white selection because the lacZα reading frame is reestablished upon religation of the vector after removal of the stuffer DNA (Figure 1A). When vectors digested with Eco RV are religated the lacZα reading frame is restored thus rendering the cells blue, whereas vectors digested with Xcm I can only yield blue colonies if both T-overhangs are lost prior to religation.

For stuffer DNA, a fragment of the yeast URA3 gene was amplified containing Eco RV and Xcm I sites for amplicon ligation and Sac I and Kpn I for cloning into pBlueScript® II KS digested with the same enzymes (Figure 1A). A selected clone was digested with different enzymes to confirm the presence of the stuffer DNA: Eco RV (558 bp), Sac I + Kpn I (570 bp), Xcm I (534 bp) (Figure 1B). The resulting vector was named pPCV (Figure 1C). This vector was digested either with Xcm I or Eco RV and the ~2.9 kb versions of the linearized vectors were named pPCV-T and pPCV-B, respectively (Figure 1C).

To test the efficiency of the resulting vectors, a yeast LEU2 gene fragment was amplified by using Phusion or Taq polymerase and the resulting amplicons ( ~1.4 kb) were ligated into pPCV-B and pPCV-T, respectively. The results of bacterial transformation are presented on Table 1 and the presence of inserts was assessed by PCR using primers 5-leud and 3-leud (Figure 2). The low percentage of white colonies observed when the pPCV-B system was used is explained by the fact that ligation of blunt-ended molecules is generally more difficult than sticky-ends. Nonetheless, a high percentage (83.3%) of white colonies had inserts. As for the pPCV-T system, most of the white colonies (90.0%) observed had inserts. All other false positives can be explained by the loss of one T-overhang following religation, which results in the loss of original lacZα reading frame as has been previously observed [Arashi-Heese et al. 1999.

The results shown in this work show that pPCV can be successfully used for high efficiency cloning of amplicons. It provides in the same cloning platform two important advantages: i) the ability to clone PCR products derived from different DNA polymerases still allowing blue/white selection and, ii) its minimal polylinker prevents undesirable restriction sites at the ends of cloned amplicon after subcloning. Plasmid pPCV is available upon request.

CRJ and ANPB carried out all the experiments described in this study as part of their MSc thesis and undergraduate training, respectively. VCBR, LMPP and FAGT acted as mentors during different stages of the project. All authors have read and approved the final manuscript.