LIC is commonly performed with T4 DNA Polymerase, which is used to generate single-stranded DNA overhangs, >12 nucleotides long, onto both the linearized vector DNA and the insert to be cloned (35). In these cases, the use of another cloning method, such as, SLIC is a standardized method for multi-fragment DNA assembly, and its low cost makes it ideal for researchers doing large amounts of cloning. Joined fragments have 4 nicks that are repaired byE.coli during transformation. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. One of the first ligation independent cloning methods was the univector plasmid fusion system, which is based on Cre/loxP-mediated recombination. 2007 Mar;4(3):251-6. As many as five inserts can be assembled in one reaction simultaneously with great efficiency using SLIC. A number of commercial cloning kits make use of site-specific recombination, requiring special vectors and costly proprietary enzymes, such as the Gateway system (Life Technologies). In the second step, the addition of a single dNTP stops the exonuclease reaction. Ligation-independent cloning is a form of molecular cloning that is able to be performed without the use of restriction endonucleases or DNA ligase. Another potential issue is sequence similarity. In SLIC, purified PCR products are treated with T4 DNA polymerase (DNAP) so that the exonuclease activity will increase the proportion of recessed ends. Please enable it to take advantage of the complete set of features! OEC uses linear amplification, generating less product than the exponential amplification in PCR. Ligation Independent Cloning (LIC) is a technique developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. Clipboard, Search History, and several other advanced features are temporarily unavailable. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. Ligation Independent Cloning (LIC) is a technique developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. Epub 2023 Jun 8. Funding: This work was supported by the National Health and Medical Research Council (1008081, www.nhmrc.gov.au/); and the National Heart Foundation of Australia (G11S5757, http://www.heartfoundation.org.au). different gap sizes) with the same 2.2 kb insert. For a convenient volume of T4 polymerase (0.75 U/0.25 L), the highest efficiency was observed after 510 min treatment at 25C, although 5 min was most robust (Table S7 in File S1), followed by immediate incubation on ice to halt the reaction. In this case, 15 bp of homologous sequence is used, plus a minimum of 18 bp of your template sequence. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. This is likely due to the increasing propensity of larger products to anneal to themselves rather than to the plasmid template. Set up a vector only control with water instead of the insert. How do I prepare and deposit my plasmids? Please enable it to take advantage of the complete set of features! It contains resistance cassettes for ampicillin and kanamycin, as well as a polylinker encoding the lacZ fragment. Accessibility The site is secure. Use 1-2 l of annealing reaction for each transformation, following a standard protocol. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. Disclaimer. 2007 Mar;4(3):251-6. Brown Construction of New Ligation-Independent Cloning Vectors for the Expression and Purification of Recombinant Proteins in Silkworms Using BmNPV Bacmid System Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. This reaction takes place in one step rather than two steps required for SLIC, and ligase may improve the efficiency of multipart assembly. (C) Colonies from ampicillin plates were patched onto kanamycin/X-gal plates to distinguish recombinants from unwanted insert vector or empty pUC18/Kan background colonies. Ligation Independent Cloning (LIC) is an alternative to restriction enzyme/ligase cloning and ensures high cloning efficiencies of more than 95% The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on vector and DNA insert. This can require the design of new primers with longer complementary tail sequences than used for restriction cloning, but the time savings and increased robustness outweigh these nominal costs, even for routine applications. Shukla PK, Radmall KS, Chandrasekharan MB. 1. The standard insert primer design was used for OEC, with annealing and 3 end-filling of partially overlapping primers alone to first prepare the insert megaprimer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes Frank Thieme, Carola Engler, Romy Kandzia, Sylvestre Marillonnet x Published: June 2, 2011 https://doi.org/10.1371/journal.pone.0020556 Article Authors Metrics Comments Media Coverage Reader Comments (0) Figures You may not be able to create an account or request plasmids through this website until you upgrade your browser. Systems, Research Editing, Cloning These are analogous to the much shorter sticky ends generated by restriction enzymes. Biotechnol Adv. National Library of Medicine Andrew J. Receive the latest news, hot plasmids, discounts and more. Copyright: 2013 Stevenson et al. To overcome these limitations, we have adopted modified ligation-independent cloning (LIC; Aslanidis and de Jong, 1990; Dieckman et al., 2002) as an approach for high-throughput cloning into the TRV VIGS vector. In our case, dGTP will be used in the reaction, meaning that T4 Pol will remove bases from the 3' end of the cut site until the first G is reached (shown in blue), at which point it will add back the G and become stalled. German chemistry CRO for global pharma, biotech & chemical companies since 1999! Ligation-Independent Cloning Tobacco Rattle Virus Vector for High T4 polymerase is used to make specific 10-15 base overhangs on the target vector and the PCR product. Because the polymerase reaction is favored over the exonuclease reaction, the polymerase will add back the guanosine residue and become stalled. PIPE cloning tolerated a wide range of insertvector molar ratios for a range of insert sizes (Table S6 in File S1), with an optimum of 2.51, similar to previously reported values for SLIC [2], [5]. Gene accession numbers and plasmid vector backbones are listed in Table S2 in File S1. 2007 Mar;4(3):251-6. doi: 10.1038/nmeth1010. 5' and 3' primers will have different leader sequences, but operate on the same principle (homologous to the first G on 3'-5' strand from cut site). We have previously observed higher efficiencies when replacing genes rather than PIPE cloning into an empty vector [6]. After the overlap is generated, dCTP is added back to the reaction, shifting the enzyme back into a polymerase, where it stalls due to the lack of a complete set of dNTPs in the buffer. Ten white colonies for each were screened by PCR across the cloning junctions. Julian Stevenson, However, cloning efficiency and robustness for OEC decrease with increasing insert size, introducing the risk of failure for larger genes. 2017 Sep 1;63(3):125-130. doi: 10.2144/000114588. The simplest effective method to reduce this background for PIPE is to use less template. SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol. We reasoned that reducing the template should dilute out the background since the complete vector is linearly amplified, it is more dependent on template concentration and thus a lower template concentration would favour PCR amplification of the desired product. 2023 May;13(5):220369. doi: 10.1098/rsob.220369. -, Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Dynarrestin, a Novel Inhibitor of Cytoplasmic Dynein - PubMed Overall, PIPE achieved cloning efficiencies of 95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. With molecular cloning scientists can amplify and manipulate genes of interest and then insert them into plasmids for replication and protein expression. This site needs JavaScript to work properly. This PCR fragment and linearized plasmid are bothpartially digested using T4 polymerase in the absence of dNTPs. The 24 bp insertion is a special case due to its small size, in that PIPE and SLIC used a single primer pair of reverse design for PCR with 3 ends complementary to the vector and 5-tails containing the entire insert thus requiring only one PCR, such that the product anneals to itself to form a circular product. The site is secure. Nevertheless, these situations can generate nicked copies of the vector in vitro that will escape DpnI digestion (Figure 2A). Brown MO, Olagunju BO, Giner JL, Welander PV. Ligation independent cloning (LIC) as a rapid route to families of Inserts are usually PCR amplified and vectors are made linear either by restriction enzyme digestion or by PCR. Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation step of traditional cloning methods. Methods Mol Biol. To test this, we replaced 350 bp, 1.4 kb, or 4.3 kb inserts in pUC18/Kan (i.e. We adopted this latter strategy in subsequent experiments. Zhang H, Liu CJ, Jiang H, Zhou L, Li WY, Zhu LY, Wu L, Meng E, Zhang DY. Step 3: Create Vector Overhangs Treat the linearized vector with T4 DNA polymerase to "chew back" the free 3' ends, following the manufacturer's instructions. It is very important to remove all free nucleotides from your PCR product before proceeding, as they will interfere with the exonuclease activity of T4 Pol in the following step. This was performed with or without linearising the vector using PstI. official website and that any information you provide is encrypted Figure 2. In both these techniques, by amplifying vector and insert with primers containing complementary 5-tails and mixing the products, the overhangs can anneal and are joined. Ligation-independent cloning of PCR products (LIC-PCR) We found that both increasing the gap and linearisation reduced background (Fig 2C). Principles of polymerase incomplete primer, Figure 1. An official website of the United States government. 0.025 pmol of vector and 0.0625 pmol insert purified products (2.51 insertvector ratio) were digested for 3 hr at 37C with 10 U of DpnI in 10 L CutSmart Buffer (NEBuffer 4.1/0.1 mg/mL BSA), diluted 11 with 1 HF buffer (to control for buffer composition between purified and unpurified products, as 1 HF buffer halved transformation efficiency), and 2 L used to transform 18 L of XL10 Gold ultracompetent cells (Agilent Technologies) according to manufacturer's instructions. If fragments in a multicomponent assembly have 5 or 3 sequence homology to each other, they may be assembled incorrectly. Epub 2007 Feb 11. We observed that PIPE worked very well with limited manipulations, as long as the template concentration was kept to a minimum to avoid overlap extension vector background. But this didn't yield any positive colonies after DpnI digestion and transformation. Unauthorized use of these marks is strictly prohibited. Ten white colonies for each were screened by PCR across the cloning junctions. PDF Arno Behr Thomas Seidensticker Chemistry of Renewables - LibManual.Com Nat Commun. How can I track requests for my plasmids? government site. However, even low concentrations of primer-dimer are of concern for OEC, since cloning is extremely efficient for very small inserts, which clone preferentially. We archive and distribute high quality plasmids from your colleagues. Ligation-independent cloning (LIC) was first developed in the 1990s. The LIC techniques tested in this study can be highly efficient and technically simple, but can be compromised by problems such as nicked vector plasmid background, cloning of primer-dimers and amplification of non-specific PCR products. -, Unger T, Jacobovitch Y, Dantes A, Bernheim R, Peleg Y (2010) Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. For all techniques, a DpnI digestion step is included to remove plasmid template (not shown). Proteins 71: 982994. We found that PIPE, SLIC and OEC were also very efficient for site-directed mutagenesis. James R. Krycer, * E-mail: aj.brown@unsw.edu.au (AJB); j.stevenson@unsw.edu.au (JS); j.krycer@garvan.org.au (JRK), Affiliation: Nat Methods. GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant We believe that LIC methods offer a more robust, efficient means for DNA cloning. It was also very vulnerable to the presence of primer-dimers. To avoid the requirement for gel extraction or PCR optimisation, alternative primer design can be used for Quick and Clean Cloning [9], a more specific variant of SLIC. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). C Aslanidis and P J de Jong Author information Copyright and License information Disclaimer Abstract A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. SLIC uses an exonuclease, T4 DNA polymerase, to generate single-stranded DNA overhangs in insert and vector sequences. However, these can be overcome through careful design, checking PCR products, and taking additional measures accordingly. Plasmids 101, doi: 10.1371/journal.pone.0153158. Instead of using T4 DNA polymerase, Gibson assembly requires T5 exonuclease in combination with Phusion polymerase and DNA ligase. Bookshelf Traditional cloning makes use of restriction enzymes and ligation of DNA in vitro. One of these interesting approaches is the use of nicking DNA endonucleases (NiDE) to create the complementary overlaps used to anneal the vector and insert. Analyzed the data: JS JRK LP AJB. A typical T4 Pol reaction is shown. HHS Vulnerability Disclosure, Help 2013 Dec;31(8):1707-21. doi: 10.1016/j.biotechadv.2013.08.021. 3 ng). Primer-dimers and non-specific products are only likely to be a problem if visible on the agarose gel or with a Bioanalyzer. Elledge realized that he could generateimperfect recombination intermediates through PCR and imprecise T4 exonuclease activity, overcoming the requirementforcarefully designed DNA overhangsused in LIC. Biosci Rep. 2017 Mar 2;37(2):BSR20160608. See main text for details. OEC yielded very high numbers of transformants for the 24 bp (84 bp megaprimer) insertion, with close to 100% efficiency. This yields a nicked plasmid that is repaired after transformation. LIC employs long overhangs to form a stable association between fragments, allowing for transformation without ligation. Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. Below we use pNIC28-Bsa4 as an example of LIC experimental design. 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. Li MZ, Elledge SJ. When working with larger amounts of DNA (~100 ng,) RecA is not required. As long as there was enough sequence homology (20-60 bp) to organize the fragments and hold them together, E. coli would be able to repair the plasmid, generating recombinant DNA. For one replicate experiment of each set, colonies were also tested by colony PCR across the cloning junctions or sequenced to validate the blue/white screening. Schwanke H, Gonalves Magalhes V, Schmelz S, Wyler E, Hennig T, Gnther T, Grundhoff A, Dlken L, Landthaler M, van Ham M, Jnsch L, Bssow K, van den Heuvel J, Blankenfeldt W, Friedel CC, Erhard F, Brinkmann MM. In E. coli, a robust homologous recombination system allows for the repair of gaps and overhangs based on regions of sequence homology. Biotechniques. Before international site. 2023 Jan 19;12:e82843. Non-specific PCR products and primer-dimers can be an important problem. Ligation independent cloning (LIC), as its name implies, allows for the joining of DNA molecules in the absence of DNA ligase. Would you like email updates of new search results? The Cytomegalovirus M35 Protein Directly Binds to the Interferon- Enhancer and Modulates Transcription of. Primer sequences are listed in Table S1 in File S1. After an hour or so, the sample is immediately ready to transform into competent cells. Ligation-independent cloning of PCR products (LIC-PCR) A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Discover a faster, simpler path to publishing in a high-quality journal. These overhangs must be accessible to allow for complementary base pairing, so SLIC cant be used if the overhangs would have stable ssDNA secondary structure. These fragments are then assembled in vitro and transformed into Escherichia coli to generate recombinant DNA of interest. Polishing the craft of genetic diversity creation in directed evolution. Ligation Independent Cloning (LIC)is a fast and easy method for cloning that doesn't use restriction enzymes or DNA ligase. 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. FOIA Show more Show more 1.4 kb Insig-1 and 4.3 kb SCAP fragments were obtained with primers T7-pUC18-F and BGH-pUC18-R from pCMV-Insig-1-Myc or pCMV-SCAP respectively [8]. . PIPE relies on incomplete extension in PCR. Supervised the project: AJB. 2. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Li MZ, Elledge SJ. The amount of DNA (25 fmol vector and 62.5 fmol insert) used for transformation was equivalent to that of unpurified PIPE cloning from a single pair of PCRs. Only one type of dNTP would be present in the reaction mix, limiting the exonuclease activity to the first occurrence of that nucleotide. Protein Expr Purif. J Struct Biol 172: 3444. Learn about the latest plasmid technologies and research tools. 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. Addition of T4 DNA polymerase exonuclease treatment for SLIC increased the number of transformants by 4100 fold for all inserts, retaining the high efficiency. One common example is the stem-loop structure of transcriptional terminators. Technique selection flowchart for a new cloning project. 8600 Rockville Pike Assembly is scarless, unlike Gateway cloning, and the methods flexibility allows it to be used with different types of PCR-generated inserts. A ligation and restriction enzyme independent cloning technique: an The 3-terminal sequence can be removed via the . , a robust homologous recombination system allows for the repair of gaps and overhangs based on regions of sequence homology. The vector and insert are combined by mixing the two products together in the absence of ligase. The annealed but nicked vector product is then repaired during the replication cycle. Ligation (molecular biology) We describe here a method for sequence- and ligation-independent cloning (SLIC). The major advantage of SLIC over Gibson assembly is cost, as T4 polymerase is much less expensive than the enzymes required for Gibson assembly. Since these products contain 5 ends complementary to the vector, they may be incorporated into recombinant clones, also yielding white colonies in our screening assay. There is a problem with the plasmid I received. Ligation-independent cloning (LIC) is a form of molecular cloning that is able to be performed without the use of restriction endonucleases or DNA ligase. PLOS ONE promises fair, rigorous peer review, [1] This allows genes that have restriction sites to be cloned without worry of chopping . Increasing the number of cycles of overlap extension gave progressively more colonies, without clearly increasing cloning efficiency (Table S10 in File S1). Figure 4. Another potential issue is sequence similarity. Please note: Your browser does not support the features used on Addgene's website. SLIC is a standardized method for multi-fragment DNA assembly, and its low cost makes it ideal for researchers doing large amounts of cloning. Each pair of insert-specific primers had the same 30 bp 5-tail this modular design allowed replacement of the multiple-cloning site of pUC18/Kan with a collection of inserts. School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, Australia, Sequence- and ligation-independent cloning: A SLIC makeover, In 2007, LIC received an important update, courtesy of Addgene depositor, . SLICs limitations arise from its dependence on single-stranded overhangs. Figure 3. The technique was developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. By harnessing the power of DNA repair in E. coli, you can assemble multiple fragments without the need for specific restriction sites or DNA ligase! BsaI), which cut at a specified distance from their recognition sequence. Well done to the authors for win-ning the richly deserved prize and to you for reading the book! Megaprimer and primer-dimer contaminant or 1.4 kb LXR megaprimer alone were gel purified and used for OEC. (2012) One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site. SLIC consistently achieved the highest efficiencies and number of transformants, but required additional resources. A variant of the OEC strategy is utilised by the commonly used QuikChange site-directed mutagenesis kit (Agilent Technologies). 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, . SLIC: a method for sequence- and ligation-independent cloning Purification of the insert PCR is recommended for OEC, but for smaller inserts, a wide range of megaprimer concentrations are tolerated, safely allowing use of a small fraction of unpurified PCR product as megaprimer (Table S12 in File S1 and data not shown). Two PCR products are used to generate a target gene with a 5 or 3 overhang. 1. The precise assembly of specific DNA sequences is a critical technique in molecular biology. A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. 9 Technische Universitt Dresden, DFG-Research Center for Regenerative Therapies Dresden, Dresden, Saxony 01307, Germany. and transmitted securely. Ligation-independent cloning mediates the assembly between a DNA fragment and a PCR-amplified vector with a 12-nt tail complementary to the DNA fragment's end. Moderate to high efficiencies were also observed for the 350 bp insertion. As such, the use of LIC is often limited to specially-designed plasmids. In contrast, OEC has two rounds of amplification. Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost. National Library of Medicine If PIPE cloning fails or a greater number of transformants are required, such as for library construction, then SLIC is superior, typically increasing the number of transformants by more than 5-fold in one step after purification, DpnI and T4 exonuclease treatment. MeSH We maintained subsequent reactions at 35 cycles to ensure that good product yields are achieved, particularly for difficult templates or inefficient primers. As long as there was enough sequence homology (20-60 bp) to organize the fragments and hold them together. The pUC18/Kan reporter vector (Figure 1B) was prepared using PIPE cloning. To more efficiently clone DNA molecules, several ligation-independent cloning (LIC) methods have been developed, such as LIC based on T4 DNA polymerase , GATEWAY recombination (6,7), In-Fusion , uracil-DNA glycosylase (UDG) , and sequence- and ligation-independent cloning (SLIC) . SLIC circumvents sequence constraints for recombinant DNA using standard restriction enzyme-mediated cloning and previous ligation-independent cloning methods and provides a new approach for the efficient generation of recombinant DNA. -, Jeong JY, Yim HS, Ryu JY, Lee HS, Lee JH, et al. SLIC uses an exonuclease, T4 DNA polymerase, to generate single-stranded DNA overhangs in insert and vector sequences. Key to SLIC is the power of homologous recombination. Inactivate T4 Pol by heating to 75 for 20 minutes. The primer length is dependent on the T4 Pol "chew back" reaction, in which a single dNTP is included to stop the exonuclease function of the enzyme and shift its activity back to polymerase.