Abstract: This study highlights a reporter-less direct-cloning protocol based on PCR and digestion/ligation reactions. Few manipulations were done allowing the user to reduce the time required for screening the right colony of any transformed cells. The obtained result showed high efficiency compared to the other commercially-available high throughput techniques, especially Gateway.
INTRODUCTION
There is no doubt that the recent cloning systems considered as a revolution in the molecular science. Such a technological development comes with two main benefits. First, it allows efficient accomplishment of complex cloning protocols in a short period of time for scientists who are already aware of molecular techniques. Second, even non-molecular scientists with low experience in molecular technology can conduct genetic modifications without any hurdles (Marsischky and LaBaer, 2004). The history of the current commercially-available cloning systems was initiated by the homologous recombination like gap-repair cloning in the yeast Saccharomyces cerevisiae (Martzen et al., 1999; Uetz et al., 2000; Datsenko and Wanner, 2000; Zhang et al., 2000). After that discovery, several disadvantages were realized which led to further advancement of a new site-specific recombination-based technology (Ito et al., 2001; Zhu et al., 2001). The site specific recombination technology has many advantages including construction of large ORF clones beside general flexibility (Liu et al., 1998, 2000, 2003; Reboul et al., 2001; Walhout et al., 2000).
However, still there are unobserved aspects to those types of technologies, such as flexibility of the vector-backbone manipulation and the total costs per reaction.
Currently, it is known that the main problem of the reporter-less direct cloning protocol of yeast is screening the transformed colonies for the right insertion. Thus, in the current study TLA polymerase, two restriction enzymes and lmbda exonuclease have been used in order to shortage the yeast classical cloning method by overcomes the bacterial cloning step and directly obtaining the right colony with the target insertion.
MATERIALS AND METHODS
Construction of [CaMV35S+SV40 T antigen] cassette: Overlapping PCR with the CaMV 35S promoter and the SV40 T antigen were used to construct the DNA cassette CaMV35S+T-antigen (Fig. 1a). Primers on the cassette’s flanks were containing restriction sites SacI and ApaI for double digestion (Table 1).
| Fig. 1(a-b): | (a) Construction of the insert-cassette with primer locations flanked by SacI and ApaI restriction sites and (b) Primer designed to linearize the plasmid vector pAcGFP-Hyg-N1 vector excluding the CMV promoter |
| Table 1: | Primers used in the current protocol |
All PCR reactions were carried out using the high fidelity TLA polymerase (Bioneer Inc., Daejeon, Korea). The PCR program was denaturation at 94°C for 10 sec, at 60°C for 20 sec, extension at 72°C for 2 min, for a total of 15 cycles.
Linearization of the pACGFP1-Hyg-N1 vector excluding the CMV promoter: Linearization and modification of the pAcGFP1-Hyg-N1 plasmid vector (Clontech) was done using TLA polymerase. Two primers were designed to start from the MCS region excluding the CMV promoter (Fig. 1b). The primers were containing the restriction sites SacI and ApaI for double digestion reaction (Table 1). The PCR carried out as follows denaturation at 95°C for 30 sec, annealing/extension at 68°C for 4 min, for a total of 15 cycles.
Digestion/ligation reaction for the linearized vector and the DNA cassette: Two endo-nuclease digestion reactions were carried out using the two enzymes SacI and ApaI according to NEB double-digest finder. The first digestion reaction was performed in two tubes by separately digesting each fragment alone. The second digestion was performed by gathering a ratio 1:3 from the linear vector: cassette insertion into one tube. After purification, fragments were ligated using T4-DNA ligase (Bioneer Inc., Daejeon, Korea).
| Table 2: | Stains and plasmids used in the current protocol |
Ligation confirmation by PCR: About 4 μL from the non-purified ligation product was used in PCR with two flank primers to confirm the ligation process (Table 1).
Purifying the circular vectors from the ligation mixture: After ligation and without purifying the ligation product, digestion reaction was carried out in a 25 μL reaction volume using lambda exonuclease for 1.5 h to start digesting the 5’end of the positive strand of the empty linear vector.
Escherichia coli transformation and colony PCR: Hit competent cells, been used with 10 μL of the non-purified product of the exonuclease digestion reaction (Table 2). Transformation was done through heat-shock. Transformation followed by refreshing the cells for two hours in liquid medium, then plating on LB medium supported with 50 μg mL–1 ampicillin. Colony PCR was done after 24 h to screen for the right insertion. Five colonies were selected randomly for colony PCR as described previously (Osek, 2001).
Yeast transformation colony PCR: In this step the purification of the Exonuclease digestion product took place using PCR purification kit (Bioneer Inc., Daejeon, Korea). Followed by electroporation of Saccharomyces cerevisiae, according to Delorme (1989) and Gunn et al. (1995). Transformation followed by cell recovery for 2 h in YPD liquid-medium, then plating on YPD plate supported with 50 μg mL–1 hygromycin. Colony PCR was done after 48 h to screen for the right insertion. Five colonies were selected randomly for colony PCR as described previously (Mirhendi et al., 2007; Luo and Mitchell, 2002) (Table 2).
RESULTS AND DISCUSSION
In regards to cassette and vector construction, increasing the template concentration resulted in some smear with some non-specific bands. However, the right bands were in much higher concentrations compared to the others (Fig. 2a). After double digestion, the ligation was confirmed using flank primers to obtain whole fragment insertion with 30 bp from the two vector’s flank-regions (Fig. 2b).
Colony PCR showed the right insertion in all colonies of E. coli culture and yeast culture as well (Fig. 2c-d). According to the screening, there was no colonies appeared without the right insertion, except for the microsatellite colonies which are usually appears smaller and beside the long-term growing recombinant colonies.
| Fig. 2(a-d): | Gel agarose showing, (a) Amplified fragments "vector and cassette" with SacI and ApaI restriction sites. M: 1kb DNA ladder. Lane C1 and C2: SV40-T antigen’s cassettes flanked by SacI and ApaI restriction sites. Lane: V1 and V2, are linear vector flanked by SacI and ApaI restriction sites, (b) Amplified fragment of T-antigen for ligation confirmation of the insertion within linear vector. M: 1 kb DNA ladder Bioneer. Lane 1: Confirmed product using the separately digested vector and cassette. Lane 2: Confirmed product using the gathered digested vector and cassette, (c) Colony PCR products for the five E. coli colonies that appeared on the LB plate without lambda Exonuclease treatment and (d) Colony PCR products for the five yeast colonies that appeared on the YPD plate with lambda exonuclease treatment |
The purpose of the current study is to highlight the equal efficiency of both the classical cloning- and high-throughput techniques (Esposito et al., 2009; Walhout et al., 2000).
The first key-point of the current protocol is eliminating the vector self-ligation problem, which usually appears in the restriction enzyme-cloning technique (Costa et al., 1994; Upcroft and Healey, 1987; You et al., 2012). Avoiding the later problem was done by linearizing the plasmid vector using non-phosphorylated primers and high-fidelity DNA-polymerase.
Although, the current experiment was done based on the sticky-end ligation, however same experiment could be done on the basis of the blunt-end ligation by simply 5’ phosphorylating the ends of the insertion DNA, besides elongating the ligation period.
Exonuclease has been reported to purify circular DNA by degrading double stranded linear-DNA (Balagurumoorthy et al., 2008; Demple and Harrison, 1994; Mol et al., 1994). In the current protocol, lambda exonuclease was used to purify circular plasmids via degrading all linear DNAs which remained in the sample after ligation including the linear empty vector.
| Table 3: | Comparison between the current protocol and Gateway cloning system |
The usage of the exonuclease in the current protocol is unnecessary with E. coli, in fact the only purpose of the circular DNA-purification step is to void any possibility of linear-heterogonous DNA recombination with any kind of recA-mutant cells. Advantages and disadvantages determined as the most important factors required by the user are shown in Table 3.
CONCLUSION
The current protocol shows the classical cloning system as a highly efficient, low-cost cloning technique as the other high-throughput systems. We focused on comparing the provided method with the Gateway cloning product by Invitrogen.
ACKNOWLEDGMENT
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0013600).