If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. Not only does this system not use site-specific recombination, it also doesn’t require a ligation step. Based on the robust system of homologous recombination found in E. coli, SLIC is a cheap, standardized, and rapid multi-part DNA assembly method. Read on to learn how to use it in your research!
The starting point: Ligation-independent cloning
Ligation-independent cloning (LIC) was first developed in the 1990s. While traditional restriction enzyme cloning uses short sticky ends to insert genes into plasmid backbones, base pairing between these ~4 base overhangs isn’t strong enough to hold a plasmid together through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs can hold a nicked plasmid together long enough to get it into cells and let them do the repair work.
The key is T4 DNA polymerase, which has 3′→5′ exonuclease activity. T4’s polymerase activity can be thwarted if no dNTPs are available to polymerize, allowing the slower exonuclease activity to proceed. By including only one type of dNTP in the reaction mix, the researcher can decide exactly how far the T4 exonuclease proceeds: at the first occurrence of the chosen nucleotide, T4 will switch to polymerizing and then stall due to the absence of the other dNTPs. Once the vector and insert are (separately) digested to create 10–12 base “chewed back” overhangs, they can be annealed, forming a circular product with four nicks easily repairable by the bacteria after transformation.
LIC is a reliable cloning method, but it is limited by its sequence constraints. The long overhangs must not contain the dNTP present in the exonuclease reaction or polymerization will occur at that position, preventing T4 from chewing back the entire 10–12 bases. As such, the use of LIC is often limited to specially designed plasmids.
Sequence- and ligation-independent cloning: A SLIC makeover
In 2007, LIC received an important update, courtesy of Addgene depositor Stephen Elledge. His new method, named sequence- and ligation-independent cloning (SLIC), eliminated many of LIC’s constraints by relying on the power of homologous recombination.
SLIC inserts and linearized vectors must have homologous DNA sequences (20–60 bp) on either end and single-stranded 5′ overhangs… but it doesn’t really matter if those overhangs perfectly match up. They just need enough complementary ssDNA to hold the plasmid together, even if there are gaps or flaps remaining. This incomplete plasmid looks like a homologous recombination intermediate to bacteria, so it can be transformed as-is into cells. The cells then get to work fixing up the “damage” and producing a complete plasmid.
Besides being compatible with any vector, the SLIC protocol itself is flexible. For one thing, those 5′ overhangs can be generated in several ways. One option, imprecise T4 digestion (no dNTPs needed), allows both insert and vector to be digested in the same tube. Alternatively, SLIC can also work directly on PCR products, with 5′ overhangs generated either by annealing a mixture of products or from incomplete PCR — although higher DNA concentrations are needed for this method to be successful.
Low DNA concentration? No problem: SLIC optionally boosts efficiency with the homologous recombination protein RecA. (It’s effective with as little as 3 ng of DNA!)
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| Figure 1: Schematic of SLIC. To start the SLIC cloning process, a gene of interest is PCR amplified to add 5′ and 3′ homology regions from the target vector. 5′ overhangs can be generated by T4 polymerase (left branch); by annealing two separate PCR products (middle branch); or by incomplete PCR, which skips the final extension step to retain products with single-stranded overhangs (right branch). The mixed PCR and incomplete PCR methods yield a minority of usable product with two 5′ overhangs, so these approaches require higher concentrations of insert to succeed. 5′ overhangs are generated in the linearized vector by T4 digestion; then the components are combined and annealed. The resulting recombination intermediate won’t just have nicks: it may have gaps or overlapping regions of single-stranded DNA. But as long as the double-stranded region is long enough to hold it together, it can be transformed into bacteria, which will efficiently repair the plasmid. Created with BioRender.com. |
SLIC also opened up a new world of multicomponent assembly, as fragments with overlapping sequence homology can be assembled in a unique order with no cloning scars. The Elledge Lab showed that a five-piece assembly reaction was highly efficient (~80%) with 40-bp homology regions. Ten-fragment assembly was also successful, but at a lower efficiency (~20%).
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| Figure 2: Schematic of multicomponent assembly with SLIC. Multiple fragments are PCR amplified to add 5′ and 3′ homology regions to the adjacent fragment or vector. 5′ overhangs are generated on all the fragments and the vector, specifying the order of assembly, and all products are annealed. The recombination intermediate is transformed into bacteria, which repair the plasmid. Created with BioRender.com. |
How does SLIC compare to other cloning methods?
SLIC’s limitations arise from its dependence on single-stranded overhangs. These overhangs must be accessible to allow for complementary base pairing, so SLIC can’t be used if the overhangs would have stable ssDNA secondary structure — for example, the stem-loop structure of transcriptional terminators.
Another potential issue is sequence similarity. If fragments in a multicomponent assembly have 5′ or 3′ sequence homology to each other, they may be assembled incorrectly. To overcome this limitation, one option is to perform a hierarchical assembly, assembling fragments in multiple steps to avoid using multiple fragments that share homology in the same reaction. In these cases, the use of another cloning method, such as Golden Gate assembly, may also be beneficial.
SLIC is most often compared to Gibson assembly, a cloning method also based on homologous recombination. The higher temperature at which Gibson assembly takes place may limit formation of secondary structures at the ends of fragments. The major advantage of SLIC over Gibson assembly is cost, as T4 polymerase is much less expensive than the enzymes required for Gibson assembly.
SLIC is a standardized method for multi-fragment DNA assembly, and its low cost makes it ideal for researchers doing large amounts of cloning. Assembly is scarless, unlike Gateway cloning, and the method’s flexibility allows it to be used with different types of PCR-generated inserts. By harnessing the power of DNA repair, you can assemble multiple fragments without the need for specific restriction sites or DNA ligase!
This post was originally written by Mary Gearing in December 2015 and was updated by Emily P. Bentley in September 2024.
References and Resources
Further reading
Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Li MZ, Elledge SJ. Nat Methods. 2007 Mar;4(3):251-6. Epub 2007 Feb 11. PubMed.
SLIC: a method for sequence- and ligation-independent cloning. Li MZ, Elledge SJ. Methods Mol Biol. 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. PubMed.
Resources at Addgene
Topics: Plasmids 101, Plasmid Cloning, Plasmids
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