![]() How does SLIC compare to other cloning methods? With 40 bp homology regions, a five piece assembly reaction is highly efficient (~80%.) Ten-fragment assembly can also be successful, but at a lower efficiency (~20%). SLIC is ideal for multicomponent assembly (see figure below), as overlapping sequence homology specifies the order of multiple fragments, and the assembly is scarless. When the products are mixed and annealed, 25% of the resulting DNA will have two single-stranded overhangs that can robustly stimulate recombination. Two PCR products are used to generate a target gene with a 5’ or 3’ overhang. Mixed PCR, shown in the middle branch of the above figure, can also be used to create an insert. If PCR does not include a final extension step, many of the products will have single-stranded overhangs due to incomplete extension, and these fragments can induce recombination. To make matters even easier, SLIC is also compatible with incompletely synthesized, or iPCR, fragments (right branch of the figure above). The PCR fragment and plasmid are then combined, annealed, and transformed into E. In the second step, the addition of a single dNTP stops the exonuclease reaction. This PCR fragment and linearized plasmid are both partially digested using T4 polymerase in the absence of dNTPs. To start the SLIC cloning process (see figure above), a fragment of interest is PCR amplified to add the specified 5’ and 3’ homology regions. When working with larger amounts of DNA (~100 ng,) RecA is not required. Adding purified RecA to the pre-transformation incubation enhances the repair process, allowing SLIC to be used with very small amounts of DNA (e.g. coli would be able to “repair” the plasmid, generating recombinant DNA. As long as there was enough sequence homology (20-60 bp) to organize the fragments and hold them together, E. Elledge realized that he could generate imperfect “recombination intermediates” through PCR and imprecise T4 exonuclease activity, overcoming the requirement for carefully designed DNA overhangs used in LIC. This process can occur through one of two pathways: RecA-mediated recombination or RecA-independent single-stranded annealing. coli, a robust homologous recombination system allows for the repair of gaps and overhangs based on regions of sequence homology. Key to SLIC is the power of homologous recombination. His new method, named sequence- and ligation-independent cloning (SLIC), eliminates many of LIC’s constraints. In 2007, LIC received an important update, courtesy of Addgene depositor Stephen Elledge. ![]() Sequence- and ligation-independent cloning: A SLIC makeover As such, the use of LIC is often limited to specially-designed plasmids. The 10-12 base overhangs must not contain the dNTP present in the reaction, or polymerization will occur at that position, preventing T4 from chewing back the entire 10-12 bases. LIC is a reliable cloning method, but it is limited by its sequence constraints. Once digested separately, the vector and insert could be annealed, forming a circular product with four nicks easily repairable by the bacteria after transformation. At that position, T4 would perform the favored polymerase reaction and subsequently stall due to the absence of other dNTPs. Only one type of dNTP would be present in the reaction mix, limiting the exonuclease activity to the first occurrence of that nucleotide. While traditional restriction enzyme cloning used short sticky ends, LIC employed the exonuclease activity of T4 DNA polymerase to create longer, “chewed-back” overhangs of about 10-12 bases. ![]() Ligation-independent cloning (LIC) was first developed in the 1990s. ![]() The starting point: Ligation-independent cloning #LIGATION INDEPENDENT CLONING SERIAL CLONER HOW TO#coli, SLIC is a cheap, standardized, and rapid multi-part DNA assembly method - read on to learn how to use it in your research. 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. If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. ![]()
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