pPCV, a versatile vector for cloning PCR products
© Janner et al.; licensee Springer. 2013
Received: 9 August 2013
Accepted: 31 August 2013
Published: 5 September 2013
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 Eco RV and Xcm I 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.
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.
Materials and methods
Strain and media
Escherichia coli XL10-Gold and DH5α were used for routine DNA manipulations. Bacterial cells were cultured in LB medium (0.5% yeast extract, 1% peptone and 1% NaCl) supplied with 100 μg/ml of ampicillin, 0.1 mM IPTG and 0.004% X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) when necessary. Genomic DNA of Saccharomyces cerevisiae S288c (MATα SUC2 mal gal2 mel flo1 flo8-1 hap1 ho bio1 bio6) [Mortimer & Johnston 1986 was used as template for amplification of the LEU2 gene.
Construction of T-vector
The stuffer DNA used in this work was derived from a fragment of the S. cerevisiae URA 3 gene present in plasmid pNKY51 [Alani et al. 1987 and was obtained by PCR using the following primers: PXCM-1 (5′-AAGGTACCGATATCTCCAATACTTGTATGGAGGGCACAGTTAAGCC-3′) and PXCM2 (5′-AAGAGCTCGATATCCTCCAATACTCCTTTGGATCCCTTCCCTTTGCAAATAGT-3′). Primer PXCM-1 contains restriction sites for Sac I, Eco RV and Xcm I while PXCM-2 has sites for Kpn I, Eco RV and Xcm I (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 MgCl2 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 Sac I and Kpn I following ligation to pBlueScript® II KS digested with the same enzymes.
To test cloning efficiency of both vectors, the S. cerevisiae LEU2 gene was cloned after amplification from yeast genomic DNA using Taq polymerase (Invitrogen) or Phusion (Finnzymes) and primers 5-leud (5′-GAGATCTATATATATTTCAAGGATATACCATTCTAATG-3′) and 3-leud (5′-GAGATCTGTTTCATGATTTTCTGTTACACC-3′). Both amplification reactions were carried out in a volume of 50 μL. For amplification with Taq polymerase, 10 ng genomic DNA was added to a reaction which included 1× PCR buffer (200 mM Tris–HCl [pH 8.4], 500 mM KCl), 2 mM MgCl2, 0.2 mM dNTP mixture, 0.2 μM each primer and 2 U Taq polymerase. The reaction was performed for 30 cycles of 94°C/45 s, 55°C/30 s, 72°C/1.5 min after an initial denaturation of 94°C/45 s. The final extension was accomplished for 10 min/72°C. The PCR system with Phusion was carried out with 10 ng genomic DNA, 1× Phusion HF buffer (1.5 mM MgCl2), 0.2 mM dNTP, 0.5 μM each primer and 0.5 U Phusion DNA polymerase. The PCR program was: 30 s at 98°C for initial denaturation following 30 cycles of 98°C/10 s, 61°C/30 s, 72°C/30 s with a final extension of 72°C/5 min. PCR products were purified as described previously and ligated into the constructed cloning vectors. Ligation was carried out in a final volume of 10 μL with a vector:insert ratio of 1:5. The system included 1 U of T4 DNA ligase (USB) and 1× reaction buffer (66 mM Tris–HCl [pH 7.6], 6.6 mM MgCl2, 10 mM DTT, 66 μM ATP), and incubation was carried out at 16°C for 16 h following transformation of E. coli DH5α cells.
Results and discussion
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).
Cloning efficiency of pPCV
% White colonies
% Recombinant clones
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.
Christiane Ribeiro Janner, Ana Lívia e Palos Brito had a fellowship from CNPq. This work was supported by FAPDF (grant 193.000.582/2009) and CNPq.
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