One-Step Sequence- and Ligation-Independent Cloning as a Rapid and Versatile Cloning Method for Functional Genomics Studies

Abstract

We developed one-step sequence- and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.

TEXT

The need for high-throughput recombinant DNA technology is rising because of the rapid increase in interest in functional genomic studies following the surge of data generated by next-generation sequencing. Construction of a library or conversion of an existing library into a different context requires a high-throughput gene cloning method, but conventional methods suffer from high cost, prolonged manipulation, or sequence restriction.

Ligation-independent cloning (LIC) is based on the 3′-to-5′ exonuclease activity of T4 DNA polymerase and has been used for 2 decades as a high-throughput method due to its uniformity and cost-effectiveness but requires a specifically designed vector containing a long stretch of sequence that lacks a particular deoxynucleoside triphosphate (1, 3–6, 9). Sequence- and ligation-independent cloning (SLIC) overcomes the sequence restraint of LIC and allows the assembly of multiple overlapping fragments simultaneously, but the cloning efficiency of SLIC in the absence of RecA is rather low (10). Various recombinase-based cloning methods, including Gateway cloning (Invitrogen), Cre-lox recombination, Red/ET recombination (Gene Bridges), In-Fusion (Clontech), Cold Fusion (System Biosciences), and CloneEZ (GenScript), have been developed, but the general use of these methods has been hampered by high cost and restrictions in the sequence or hosts (2, 12–15).

We have optimized SLIC to make it comparable to commercial methods in terms of simplicity, time saving, and cloning efficiency. One-step SLIC utilizes only T4 DNA polymerase but shows cloning efficiencies similar to those of the original SLIC method in the presence of RecA and of commercial methods. An overview of one-step SLIC is illustrated in Fig. 1A. First, the vector needs to be linearized by either restriction enzyme digestion or inverse PCR. An insert(s) is prepared by PCR with primers with a 15-bp or longer extension homologous to each end of the linearized vector. Second, the vector and insert(s) are mixed and incubated at room temperature for 2.5 min with T4 DNA polymerase to generate 5′ overhangs. For optimal results, a 1:2 to 1:4 molar vector-to-insert ratio is desirable. Third, the reaction mixture is placed on ice for 10 min for single-strand annealing and then competent Escherichia coli cells are transformed with the annealed DNA complex directly. The annealed complex turns into seamless recombinant DNA through homologous recombination in vivo with high efficiency. For a detailed description of the method, see the supplemental material.

Fig 1.

Overview of one-step SLIC. (A) Schematic diagrams of one-step SLIC. (B) Partial sequences of the vector and insert. The arrows below the sequences indicate the forward and reverse primers used to amplify the insert. Homologous regions are in the same color. The BamHI restriction site is in bold. (C) Restriction map of the vector and insert. (D) Analysis of recombinants. Plasmid DNAs purified from 22 independent colonies (numbered 1 to 22) derived from Fig. 2A (from a sample treated for 2.5 min) were digested with EcoRI and analyzed on an agarose gel. The EcoRI-digested vector used as a control (lane V) yields 4.8- and 0.1-kb fragments, while the correctly recombined clone yields 4.25-, 1.6-, and 0.1-kb fragments. Lane M, molecular size markers.

pUC118-HMG (11) was cleaved with BamHI, and a 1-kb insert containing Ton_0709 from the genomic DNA of Thermococcus onnurineus NA1 (8) was amplified by PCR with forward (blue arrow) and reverse (red arrow) primers that have 22 bp of homology to the BamHI-cleaved vector end (Fig. 1B; see Table S1 in the supplemental material). Both the linearized vector and the PCR product were purified with a commercial PCR purification kit and eluted in 10 mM Tris Cl, pH 8.5. One hundred nanograms of the vector and 40 ng of the insert were mixed with buffer 2 plus bovine serum albumin (NEB) in a 10-μl reaction mixture and treated with 0.6 U of T4 DNA polymerase (NEB) at room temperature for 2.5 min. The mixture was put on ice immediately to stop the reaction. After 10 min of annealing on ice, 1 μl of the reaction mixture was added directly to home-prepared competent cells of E. coli strain DH5α (100 μl) or TOP10 (50 μl; Invitrogen). E. coli cells were incubated on ice for 20 min, heat shocked at 42°C for 45 s, returned to ice for 2 min, combined with 0.9 or 0.95 ml of LB, and recovered at 37°C for 1 h. Ten- to 100-μl volumes of the cells were plated on 100 μg/ml ampicillin plates and incubated at 37°C for 16 h. The gaps generated by excessive exonuclease activity are repaired, and annealed strands are joined efficiently by homologous recombination in vivo (7, 9, 10). The recombination efficiency was 100% when 22 colonies were randomly picked and analyzed by EcoRI digestion (Fig. 1C and D). Incubation on ice for more than 10 min or storage at −20°C did not alter transformation efficiency, but briefer incubation on ice did result in reduced efficiency (data not shown).

In the original SLIC method, the vector and insert were treated with T4 DNA polymerase at room temperature for 30 min to 1 h in separate tubes, combined with dCTP to stop the reaction, and annealed at 37°C for 30 min (10). To optimize one-step SLIC, the time of vector and insert incubation with T4 DNA polymerase at room temperature was varied from 0 to 60 min. We found that 2.5 min is sufficient to generate 5′ overhangs for single-strand annealing and that incubation for more than 5 min severely impairs cloning efficiency (Fig. 2A). When T4 DNA polymerase was treated with various concentrations of the vector at a vector-to-insert molar ratio of 1:2, a vector concentration as low as 0.5 ng/μl gave a sufficient number of colonies (Fig. 2B). To evaluate the effect of the vector-to-insert molar ratio, we mixed the vector (100 ng) with appropriate amounts of the insert and treated the combination with T4 DNA polymerase for 2.5 min. The cloning efficiency was highest when the vector-to-insert molar ratio was 1:4 (Fig. 2C). One-step SLIC was efficient when 0.3 U or more T4 DNA polymerase was used to treat 100 ng of the vector at a vector-to-insert molar ratio of 1:2 (Fig. 2D).

Fig 2.

Optimization of one-step SLIC. (A) Effect of the duration of T4 DNA polymerase treatment on one-step SLIC. One hundred nanograms of BamHI-digested pUC118-HMG (4.9 kb) was mixed with an insert (Ton_0709, 1 kb, 22 bp of homology) at a 1:2 vector-to-insert molar ratio in a 10-μl reaction mixture. The mixture was incubated with T4 DNA polymerase (0.6 U) at 22°C for the indicated times. The reaction mixture was than incubated on ice for 10 min, and competent TOP10 cells were transformed with the annealed DNA complex. (B) Effects of different vector DNA concentrations on one-step SLIC. Various concentrations of the vector were mixed with the insert at a 1:2 vector-to-insert molar ratio. The DNA complex was treated with T4 DNA polymerase (0.6 U) at 26°C for 2.5 min. After 10 min on ice, competent DH5α cells were transformed with the annealed DNA complex. (C) Effect of the vector-to-insert molar ratio on one-step SLIC. (D) Effect of the amount of T4 DNA polymerase on one-step SLIC. Each bar represents the mean ± the standard deviation of triplicates.

To determine the minimal homology length for one-step SLIC, primers with different homology lengths were used for PCR. Cloning efficiency was proportional to the homology length, and primers with 10 bp or more homology gave sufficient numbers of colonies (more than 5 × 10⁵ CFU/μg vector) when the homologous region matched the end of the linearized vector perfectly (Fig. 3). In many cases, the endonuclease site of the insertion point in the vector needs to be deleted or switched to a different restriction site. LIC was shown to tolerate these changes (9). When 4 bp of the BamHI sequence (Fig. 3B, underlined) was deleted from the insert, the minimal homology length required for one-step SLIC increased to 15 bp (Fig. 3A).

Fig 3.

Comparison of the cloning efficiencies of inserts with a perfect match and those with a 4-bp deletion. (A) The insert (Ton_0709, 1 kb) was amplified by PCR with primers with various lengths of homology. BamHI-digested pUC118-HMG was mixed with the insert at a 1:2 molar ratio at 10 ng vector/μl and treated with T4 DNA polymerase (0.6 U) for 2.5 min, and competent TOP10 cells were transformed with the DNA complex. Each bar represents the mean ± the standard deviation of triplicates. (B) Partial sequences of the vector and insert. Homology regions are in bold, and the deleted region is underlined.

To test if multiple fragments can be cloned simultaneously by one-step SLIC, 1-kb fragments flanking Ton_1323 were amplified from T. onnurineus genomic DNA by PCR with primers that have 21 to 25 bp of homology at their ends. pUC118-del-HMG, a vector designed to construct markerless deletion mutants of T. onnurineus, was digested with SmaI and mixed with the left and right arms of Ton_1323 in various vector-to-insert molar ratios at a vector concentration of 10 ng/μl (Fig. 4A to C). The DNA was treated with T4 DNA polymerase for 2.5 min, and competent TOP10 cells were transformed with the DNA. A vector-to-insert molar ratio of 1:2:2 gave the highest number of recombinant clones (Fig. 4B). When 8 colonies from each group were randomly picked and analyzed by restriction digestion, 87.5% resulted in positive recombinant clones (Fig. 4C). To further check if four-fragment assembly is possible with this technology, the pUC118-HMG vector was digested with BamHI and PstI to produce a 3.1-kb backbone and a 1.8-kb HMG cassette. Digested fragments were copurified with a commercial PCR purification kit and mixed with left and right flanking regions of Ton_0707 at a 1:1:1:1 molar ratio of backbone to HMG cassette to left arm to right arm (Fig. 4D). Restriction digestion analysis of the colonies resulted in 95.8% correctly recombined clones (Fig. 4E and F), clearly demonstrating that one-step SLIC can also be used as a multiple-fragment assembly technique. It is worth mentioning that the HMG cassette shown in Fig. 4D is not produced by PCR, implying that multiple-fragment assembly can be used widely to construct a composite vector with multiple fusion tags or regulatory elements or to clone large cDNAs or genes in a single cloning.

Fig 4.

Simultaneous assembly of multiple fragments using one-step SLIC. (A) Schematic diagram of three-fragment assembly. (B) Cloning efficiencies of three-fragment assembly. (C) Analysis of recombinants from the three-fragment assembly with NcoI. Aberrant clones are in red. V, vector; M, molecular size markers. (D) Strategy of four-fragment assembly. The pUC118 backbone (3.1 kb) and HMG cassette (1.8 kb) were copurified after the digestion of pUC118-HMG with BamHI and PstI and mixed with the left and right flanking regions of Ton_1323 generated by PCR, and one-step SLIC was performed. (E) Cloning efficiencies of four-fragment assembly. (F) Analysis of recombinants from the four-fragment assembly with HindIII. The aberrant clone is in red. LA, left arm; RA, right arm. Each bar in panels B and E represents the mean ± the standard deviation of triplicates.

We routinely use this technology to produce gene knockout constructs for T. onnurineus as a single- or multiple-fragment assembly. Sequencing analyses revealed 100% correctly recombined clones at the junctions of the recombinant DNA even when homologous regions at the ends of the insert did not exactly match the ends of the linearized vector, as shown in Fig. 3. One-step SLIC is highly versatile, since one can insert a gene of interest into any vector at any position without additional sequences or suitable restriction site. By modulating the junction between the homologous and gene-specific regions of PCR primers for the insert, one can delete or add any additional sequences, such as a restriction enzyme site or a tag sequence (9). In case there is no restriction enzyme site available, the vector can be linearized by reverse PCR with subsequent DpnI treatment. Like LIC, one-step SLIC also offers uniform conversion of genes from one context to another regardless of sequence variations of the genes of interest since there is no need to digest inserts with restriction endonucleases, providing a robust basis for high-throughput gene cloning. Together with its cost- and time-saving properties, one-step SLIC makes high-throughput gene cloning practical for functional genomic studies. Although we have only tried the assembly of up to four fragments, we believe that the assembly of more than four fragments by one-step SLIC may be possible. This would greatly facilitate synthetic biology, as well as the cloning of long DNA sequences with multiple restriction enzyme sites.