Overlap extension cloning of PCR products into a Gateway-compatible plasmid vector

DNA cloning is a basic step in recombinant DNA technology where a DNA sequence of interest is inserted into a suitable vector, usually a plasmid. The traditional “cut-and-paste” DNA cloning techniques use restriction enzymes, DNA ligase, and other enzymatic reaction and purification steps that are time-consuming and cost-inefficient [1,2]. More recently developed restriction-free cloning techniques have significantly streamlined the procedure. Among them, PCR-mediated cloning [3–7] and recombinase-dependent cloning [3,4,8,9] have become widely established.

PCR-mediated cloning approaches allow inserting a DNA fragment into any position of a vector, maintaining the directionality of the insert, and can be performed without sequence constraints [3,5–7,10,11]. The amplicon is inserted into a specific site of the vector in the process of PCR amplification. Overlap extension PCR cloning is a type of PCR-mediated cloning approach where DNA fragment is amplified in PCR reaction using chimeric primers containing insert-specific and adapter regions [4–7,10,11]. The adapter regions share homology with sites on the target cloning vector, ensuring directionality and insertion into a specific site. Due to the homology between flanking adaptor regions and target vectors, the amplified insert acts as a megaprimer during the subsequent linear amplification reaction [3–5,10,11]. The resulting recombined DNA product is directly transformed into competent E. coli bacteria which repair single-stranded nicks in the circular DNA. This simple and efficient technique can be used for large-scale cloning projects.

The Gateway™ system (Thermo Fisher Scientific, MA, USA) is an example of recombinase-dependent cloning. It uses the bacteriophage lambda site-specific recombination to “shuttle” a gene of interest into a plasmid vector [1,12–15]. A DNA fragment can be introduced into the Gateway cloning system either by ligation into pENTR™ plasmids or by the BP recombination reaction into pDONR™ vectors. The latter approach requires adding BP recombinase recognition sites to amplification primers. The selection for recombinant plasmids is aided by removing the negative slection ccdB gene [16] present in the Gateway cassette. Although this cloning system is highly efficient, it has several limitations, including restricted choice of pENTR™ plasmids with different antibiotic selections and the high cost of proprietary recombinase enzymes and cloning vectors.

A restriction-free PCR cloning system that utilizes ccdB gene replacement and gentamicin resistance dual selection in a new entry vector pGATE-1 is introduced here. A modification of the overlap extension PCR-mediated cloning allows for efficient and cost-effective insertion of a DNA amplicon into the Gateway system that bypasses BP and ligase reactions. The applicability of the cloning technique expands beyond the Gateway system and can be used to efficiently clone any PCR product using the dual-selection system.

Materials & methods

Construction of the pGATE-1 entry vector

To expand the available suite of the Gateway system entry vectors a new entry vector, pGATE-1, that has gentamicin resistance was constructed. The TRM13 (AT2G45900) gene that was cloned previously into pDONR207 was replaced with a Gateway cassette containing the chloramphenicol resistance gene and ccdB gene. The chimeric primers used for amplification of the Gateway™ cassette from the pDONR207 were designed with the molecular sequence analysis software Vector NTI [17,18]; Thermo Fisher Scientific): ccdB attL1 F (5′-AAG TTT GTA CAA AAA AGC AGG TCG ACC GAC AGC CTT CCA AAT G-3′) and ccdB attL2 R (5′-CAC TTT GTA CAA GAA AGC TGG TCG ACT AAG TTG GCA GCA TC-3′). The primers are complementary to the Gateway cassette flanking region (shown in bold; underlined are the SalI recognition sites) and to attL1 and attL2 seqyences. The resulting 2036-base pair (bp) PCR product was used as a megaprimer (henceforth referred to as the GATE1 megaprimer) in the overlap extension step described below. Next, the pDONR207-attL vector backbone with attL sites was amplified using primers complementary to the pDONR207-TRM13 plasmid backbone pENTR-OMG V F (5′-GTC GAC CAG CTT TCT TGT ACA AAG TGG GCA TTA TAA G-3′) and pENTR-OMG V R (5′-GTC GAC CTG CTT TTT TGT ACA AAC TTG GCA TTA TAA AAA AG-3′).

To combine the resulting PCR fragments, an overlap extension cloning method was used essentially as described in previous reports [4] with minor modifications. The GATE-1 megaprimer was used as an insert and the amplified pDONR207-attL backbone was used as a template. The linear amplification took place in 10x Taq buffer using 75 ng of vector backbone with a 3:1 megaprimer-to-vector molar ratio, 0.4U Taq DNA polymerase (Midsci, MO, USA; #BETAQ-5000) and 0.5U Phusion DNA polymerase (New England BioLabs, MA, USA; #M0530L) in 30 μl total volume. The mixture was heated to 98°C for 60 s for initial denaturation, followed by 25 cycles of 10 s denaturation at 98°C, 30 s of annealing using touchdown from 65° to 54°C and 150 s of elongation at 72°C terminated by a final 120 s elongation step at 72°C. The endonuclease DpnI (10 U; New England BioLabs) was added to the reaction and left to incubate at room temperature overnight. DpnI digestion was heat-inactivated by heating to 80°C for 20 min.

The product of the overlap extension reaction was transformed into the E. coli strain DB3.1 and selected on gentamicin (10 mg/l) and chloramphenicol (25 mg/l) antibiotics. Isolated pGATE-1 plasmids were verified by restriction digestion and sequencing.

Verification of pGATE-1 functionality & improving efficiency of the overlap extension PCR cloning

To check the functionality of pGATE-1 in cloning a PCR amplified DNA fragment, primers were designed to clone the FOP gene (National Center for Biotechnology Information reference sequence: NC_000012): FOP Ins F (5′-GTT TGT ACA AAA AAG CAG GTC GACATG GCG GCG ACG GCG G-3′) and FOP Ins R (5′-CTT TGT ACA AGA AAG CTG GTC GACCTA TGC AAC ATC TTC CAG ATA ATC CGC-3′) with italicized nucleotides complementary to the pGATE-1 backbone followed by the FOP gene-specific region shown in bold. The SalI sites flanking the insert are underlined. The FOP megaprimer was amplified in the PCR-1 reaction with 5x Phusion HF buffer (New England BioLabs; #M0530L), 200 μM dNTP (Thermo Fisher Scientific), 0.5 μM of both primers FOP Ins F and FOP Ins R, 0.6U of Phusion DNA polymerase (New England BioLabs; #M0530L) and 75 ng of FOP/pCACT2 template plasmid DNA in 30 μl total volume. The samples were denatured at 98°C for 60 s, followed by 25 cycles of 10 s denaturation at 98°C, 30 s annealing at 66°C and 60 s elongation at 72°C; the final extension step was 120 s elongation at 72°C. The PCR product was used directly in the subsequent overlap extension reaction without purification. Overlap extension cloning was used to swap the Gateway cassette in pGATE-1 with the FOP megaprimer. Four different modifications of the overlap extension reaction (PCR-2) were tested to improve cloning efficiency.

Reaction 1 conditions

DNA polymerase mix with touchdown. Linear amplification took place in 10x Taq buffer (Midsci, MO, USA; #BETAQ-5000), 1.5 mM MgCl₂, 200 μM dNTP, 1.0U Taq DNA polymerase (Midsci; #BETAQ-5000) and 0.4U Phusion DNA polymerase using 25 ng of pGATE-1 vector and a 20:1 megaprimer-to-vector molar ratio. Nuclease-free water was added to bring the total reaction volume to 30 μl. The mixture was heated to 98°C for 30 s for initial denaturation, followed by 30 cycles of 10 s denaturation at 98°C, 30 s annealing using touchdown from 70° to 60°C and 150 s of elongation at 72°C. The reaction was terminated by a final 120-s elongation step at 72°C.

Reaction 2 conditions

DNA polymerase mix without touchdown. The mixture (as in Reaction 1) was heated to 98°C for 60 s for initial denaturation, followed by 30 cycles of 10 s denaturation at 98°C, 30 s annealing at 63°C and 150 s of elongation at 72°C. The reaction was terminated by a final 120-s elongation step at 72°C.

Reaction 3 conditions

Phusion DNA polymerase with touchdown. Linear amplification took place in 5x Phusion buffer using 25 ng of vector backbone with a 20:1 megaprimer-to-vector molar ratio and 0.6U Phusion DNA polymerase. Nuclease-free water was added to bring the total reaction volume to 30 μl. The PCR cycle parameters were as in Reaction 1.

Reaction 4 conditions

Phusion DNA polymerase without touchdown. The PCR mixture and cycle parameters were as in Reaction 2. The unpurified products of the overlap extension reactions (PCR-2) were used for transformation into the chemically competent DH5α E. coli strain.

 Transformed bacteria were plated on LB agar plates containing gentamicin (10 mg/l) and incubated at 37°C overnight. Individual colonies were inoculated in liquid LB media with gentamicin, and plasmids were purified using the IBI Hi-Speed Mini Plasmid Kit (IBI Scientific, IA, USA; #IB47101). FOP/pGATE-1 plasmids were tested by PCR, restriction digest and sequencing. The functionality of the FOP/pGATE-1 plasmid was verified in the Gateway LR reaction (Thermo Fisher Scientific; LR Clonase™ II) with the pCD2 destination vector. The resulting plasmids were tested by restriction digestion, confirming a successful LR reaction and the functionality of pGATE-1 as an entry vector for Gateway cloning.

Upon publication, the pGATE-1 plasmid will be made available through Addgene (https://www.addgene.org/). The finalized pGATE-1 cloning method with optimized overlap extension conditions is shown in Supplementary Figure 1.

Results & discussion

Construction of the pGATE-1 vector for cloning PCR products

The pGATE-1 plasmid is a 5313-bp entry vector designed for the introduction of PCR fragments into the Gateway cloning system. The ccdB gene allows for negative selection and the gentamicin resistance gene allows for positive selection following bacterial transformation. Most commercially available pENTR™ vectors use kanamycin selection, limiting flexibility when working with kanamycin-resistant destination vectors. The pGATE-1 vector offers additional flexibility in cloning PCR fragments into Gateway-compatible vectors.

pGATE-1 was constructed using a modification of the overlap extension PCR cloning technique involving two PCR amplification steps [5,10,11]. PCR-1 amplified the Gateway cassette of pDONR207 flanked by added attL sites. The amplicon from the PCR-1 reaction containing the ccdB cassette served as a megaprimer during the overlap extension (PCR-2) reaction. The resulting pGATE-1 entry vector contained the Gateway cassette between attL sites (Figure 1).

Insertion of DNA fragments into pGATE-1 vector by the overlap extension PCR cloning with improved efficiency

To test the functionality of pGATE-1 as a cloning vector, we used a modified overlap extension technique to introduce the FOP gene into pGATE-1 (Figure 2). In this technique, the principle of gene replacement [19] is used to swap the ccdB-containing region in pGATE-1 with the desired PCR fragment. The FOP gene was PCR-amplified with chimeric primers in which the 3′ ends were complementary to the FOP gene and the 5′ ends were complementary to the attL sites flanking the Gateway cassette in pGATE-1 (Figure 3).

To optimize the cloning procedure, we tested different parameters of the overlap extension PCR [4] resulting in a modified protocol with improved efficiency. Four different PCR modifications were used in the PCR-2 step. Reaction 1 conditions had mixed polymerases with touchdown; Reaction 2 conditions had mixed polymerases without touchdown; Reaction 3 conditions had Phusion DNA polymerase only with touchdown; and Reaction 4 conditions had Phusion DNA polymerase without touchdown. Due to the presence of the ccdB negative selection marker in the Gateway cassette, the DpnI digest was not performed either after PCR-1 nor after PCR-2

Following transformation of equal volumes of PCR-2 products, Reaction 1 conditions resulted in the highest number of bacterial colonies. There were 309 colonies for Reaction 1 and 51 colonies for Reaction 2. Reaction 3 (Phusion polymerase with touchdown) and Reaction 4 (Phusion polymerase without touchdown) produced no colonies (Table 1, Experiment 1). Ten colonies from Reaction 1 and Reaction 2 plates were used for plasmid isolation and PCR verification for the presence of the FOP gene. For both Reaction 1 and Reaction 2 conditions, all ten colonies contained plasmids with the FOP gene (Figure 4).

These findings are consistent with other reports on the touchdown method improving both specificity and yield of PCR amplification [20,21]. Although other reports successfully used overlap extension PCR cloning using Phusion polymerase [4,10], and we used it for the construction of the pGATE-1 plasmid (albeit with a low number of positive colonies), we were able to successfully clone the FOP gene into pGATE-1 only when a blend of a nonproofreading Taq and Phusion polymerase was used. A combination of two factors provides a likely explanation for this result. First, the difference in competent cells' efficiency may explain the presence of positive bacterial colonies in the other reports and their absence in this study. In our experiments, self-prepared competent cells with an efficiency of about 10⁷ CFU/μg were used, compared with 10⁹ CFU/μg efficiency typically found in commercially available competent cells. Second, the blend of a nonproofreading Taq and Phusion polymerases is likely more efficient in the PCR-2 reaction than the Phusion polymerase alone. The large size of the megaprimer (up to 2036 bp in this study) would favor the formation of the primer–primer duplex over the primer–template duplex. Noteworthy, the Phusion polymerase was reported to be inefficient in PCR reactions with primer overlapping more than 28 bp [22]. It was suggested that the intrinsic nature of different polymerases is responsible for their differential ability to use primers with varying degrees of overlap [22]. Combining nonproofreading Taq and Phusion polymerases may have increased the primer–template duplex template formation resulting in a more efficient amplification.

In a separate experiment, we compared the effect of PCR cleanup following PCR-1 on the cloning efficiency. PCR-2 was performed on two PCR-1 samples amplified using Reaction 1 conditions (mixed polymerase with touchdown) with one sample subjected to PCR cleanup (GeneJET PCR purification kit; Thermo Fisher Scientific). The PCR-1 purified sample produced 98 colonies while the unpurified sample produced 28 colonies (Table 1, Experiment 2). Ten colonies were selected from each plate and tested with PCR for the presence of the FOP gene. In both cases, ten out of ten colonies tested positive for FOP. This indicated that cloning efficiency was moderately improved by including a PCR cleanup step; however, it can be omitted to save time without sacrificing the ratio of positive cloning events.

Both Gateway and conventional ligation-mediated cloning are facilitated by using the source and target plasmids with different antibiotic resistance. The unrecombined source plasmids often cause high background, making it nearly impossible to identify recombined plasmids with a cloned fragment. Although there are different ways to circumvent this problem, they involve time-consuming enzymatic and purification steps. For example, in Gateway LR reactions using plasmids with the same resistance, the entry plasmid must be linearized with restriction digestion followed by DNA purification. Besides the time and labor involved, this approach is limited to DNA fragments that do not contain restriction sites used for the linearization of the vector backbone. Here we tested if PCR extension cloning into the pGATE-1 can be used to clone DNA fragments from plasmids with the same resistance as the target vector (gentamicin). The FOP gene was amplified from pDONR207 and inserted into pGATE-1 following the described technique. Thirteen colonies were tested with PCR to identify the presence of the FOP gene and all contained the FOP gene. DNA restriction analysis was performed to differentiate between FOP/pDONR207 and FOP/pGATE-1 plasmids. It showed that 3 out of 13 plasmids contained the FOP gene in pGATE-1 (Table 1, Experiment 3). The 23% success rate makes this technique feasible for subcloning DNA fragments into vectors with the same antibiotic resistance without additional enzymatic and purification steps.

In a functionality test, the FOP/pGATE-1 plasmid was used successfully in the Gateway LR reaction indicating that pGATE-1 can function as an entry clone in the Gateway system.

Conclusion

The combination of an improved overlap extension PCR method and novel pGATE-1 cloning vector is an efficient and cost-effective molecular cloning system for introducing DNA fragments into the Gateway cloning pipeline. It does not rely on the Gateway BP reaction for PCR product cloning, eliminating the need for the costly BP recombinase enzyme. Beyond use in the Gateway system, cloning into pGATE-1 is universally applicable to insert any PCR product into the plasmid vector simply by adding 24 bp of pGATE-1 complementary sequences to specific primers. It is a ligation-free cloning system that does not require DNA purification and plasmid backbone preparations. This highly efficient cloning system benefits from dual selection and can also be used for subcloning of DNA fragments where both source and target plasmids have the same antibiotic resistance without additional DNA manipulations.

Future perspective

This study provides researchers with a simple and cost-efficient DNA cloning technique. The described optimized overlap extension technique can be potentially adapted for Gateway destination vectors, eliminating the need and associated costs of the Gateway LR reaction. Indeed, a similar protocol was successfully used for inserting a gene into a destination vector [10]. However, this approach has limited applicability due to the larger size of destination vectors requiring more elaborate techniques such as long-distance PCR or Gibson assembly. Although the described cloning method is highly efficient, the number of positive cloning events can be further improved by using Exponential Megapriming PCR [7] at the overlap extension step. Beyond the Gateway system, the pGATE-1 plasmid and the modified overlap extension protocol can be used to clone PCR products for ligation-mediated subcloning simply by adding restriction enzyme recognition sites to PCR-1 primers between the 24-bp adaptor and gene-specific sequences. The simplicity and high efficiency of the described cloning system make it feasible in high-throughput cloning technologies.