pPCV, a versatile vector for cloning PCR products

The efficiency of PCR product cloning depends on the nature of the DNA polymerase employed because amplicons may have blunt-ends or 3′ adenosines overhangs. Therefore, for amplicon cloning, available commercial vectors are either blunt-ended or have a single 3′ overhanging thymidine. The aim of this work was to offer in a single vector the ability to clone both types of PCR products. For that purpose, a minimal polylinker was designed to include restriction sites for EcoRV and XcmI which enable direct cloning of amplicons bearing blunt-ends or A-overhangs, respectively, still offering blue/white selection. When tested, the resulting vector, pPCV, presented high efficiency cloning of both types of amplicons.


Introduction
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 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 BciVI, BfiI, HphI, MnlI, TaaI, XcmI and Eam1105I [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.

Construction of T-vector
The stuffer DNA used in this work was derived from a fragment of the S. cerevisiae URA3 gene present in plasmid pNKY51 [Alani et al. 1987] and was obtained by PCR using the following primers: PXCM-1 (5′-AAGGTACCGATAT CTCCAATACTTGTATGGAGGGCACAGTTAAGCC-3′) and PXCM2 (5′-AAGAGCTCGATATCCTCCAATACTC CTTTGGATCCCTTCCCTTTGCAAATAGT-3′). Primer PXCM-1 contains restriction sites for SacI, EcoRV and XcmI while PXCM-2 has sites for KpnI, EcoRV and XcmI (all sites are underlined). Both primers have sequences complementary to URA3 which allow amplification of a~600 pb stuffer DNA fragment. PCR was carried out in a volume of 50 μL containing 1.5 ng pNKY51, 0.2 mM dNTP, 0.2 μM each primer, 1× PCR buffer (100 mM Tris-HCl [pH 8.5], 500 mM KCl), 2 mM MgCl 2 and 2 U Taq polymerase (LCG Biotechnology). Amplification was performed for 30 cycles of 94°C/45 s, 65°C/45 s, 72°C/40 s after an initial denaturation step of 94°C/45 s. A final extension step was performed for 2 min/72°C. The resulting amplicon was purified with UltraClean PCR Clean-Up Kit (MO BIO) and digested with SacI and KpnI following ligation to pBlueScript® II KS digested with the same enzymes.

Results and discussion
For vector construction, a minimal polylinker was designed ( Figure 1A) with the inclusion of restriction sites for XcmI, which produce 3′-T overhangs that can be used for cloning PCR products derived from amplification by Taq polymerase, and EcoRV, which yields blunt-ends suitable for cloning PCR products generated by Pfu DNA polymerases. It is argued that the use of XcmI 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 EcoRV are religated the lacZα reading frame is restored thus rendering the cells blue, whereas vectors digested with XcmI 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 EcoRV and XcmI sites for amplicon ligation and SacI and KpnI 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: EcoRV (558 bp), SacI + KpnI (570 bp), XcmI (534 bp) ( Figure 1B). The resulting vector was named pPCV ( Figure 1C). This vector was digested either with XcmI or EcoRV 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.