-, Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. PLOS ONE promises fair, rigorous peer review, Taros Chemicals | 3,622 followers on LinkedIn. In our experience, these non-specific products can also be reduced through design of longer 3 ends, increasing annealing temperatures, and reducing primer concentration. How can I track requests for my plasmids? Julian Stevenson, His new method, named sequence- and ligation-independent cloning (SLIC), eliminates many of LICs constraints. For genes of up to 1.5 kb, OEC is a good choice, as useful efficiencies (50%+) can be achieved with one primer pair. When you are looking to clone with confidence, think of NEB. Andrew J. Hence, bacteria were first plated onto ampicillin plates and then patched onto kanamycin selective plates in the presence of IPTG for blue/white colony screening. By harnessing the power of DNA repair in. While restriction-ligation cloning can be slow and inefficient, ligation-independent cloning uses long single-stranded overhangs generated by T4 DNA polymerase's 3 exonuclease activity to anneal the insert and plasmid vector prior to transformation. Here, a gene of interest was cloned in the so-called pUNI vector and then transferred into a pHOST vector providing the regulatory sequences for expression [3] , which allowed standardized and quick . 40 colonies were picked and streaked onto kanamycin selective plates in the presence of X-gal/IPTG to identify recombinants. Julian Stevenson, Other variations on the basic LIC design principles have been published and put into practice in various labs. The vector and insert are combined by mixing the two products together in the absence of ligase. Reaction products were diluted with either HF buffer or NEBuffer to control for transformation buffer composition, as the Phusion 1 HF PCR buffer halved transformation efficiency (data not shown). 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. If fragments in a multicomponent assembly have 5 or 3 sequence homology to each other, they may be assembled incorrectly. Would you like email updates of new search results? These fragments are then assembled in vitro and transformed into Escherichia coli to generate recombinant DNA of interest. Ligation-independent cloning (LIC) was first developed in the 1990s. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Federal government websites often end in .gov or .mil. Accordingly, PIPE cloning efficiency increased from approximately 40% to greater than 95% when using ten-fold less template (Figure 2B), with similar results for insertion of the 350 bp, 1.4 kb and 4.3 kb inserts into pUC18/Kan (Table S3 in File S1). Ten white colonies for each were screened by PCR across the cloning junctions. We believe that LIC methods offer a more robust, efficient means for DNA cloning. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. Proteins 71: 982994. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer . This work was supported by the National Health and Medical Research Council (1008081. LIC is much faster and more accurate than other cloning strategies as only a single transformation is required. In. Restriction endonuclease digestion and ligation increase the complexity of cloning projects, for example by requiring selection of appropriate restriction-sites and inefficient ligation steps. 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. MeSH terms Base Sequence Please enable it to take advantage of the complete set of features! 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 consistently achieved the highest efficiencies and number of transformants, but required additional resources. However, to create point mutations we routinely use an alternative two-stage cycling strategy similar to OEC, adapted from QuikChange [13], [14]. Meng X, He M, Chen B, Xia P, Wang J, Zhu C, Wang H, Zhu G. Microorganisms. 2016 Apr 13;11(4):e0153158. One common example is the stem-loop structure of transcriptional terminators. Nat Methods 4: 251256. Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation step of traditional cloning methods. After the digest is complete, you will need to separate the linearized vector from the reaction mixture by gel electrophoresis followed by gel purification. With 40 bp homology regions, a five piece assembly reaction is highly efficient (~80%.) SLIC: a method for sequence- and ligation-independent cloning. SLIC uses an exonuclease, T4 DNA polymerase, to generate single-stranded DNA overhangs in insert and vector sequences. After an hour or so, the sample is immediately ready to transform into competent cells. This yields a nicked plasmid that is repaired after transformation. Ligation Independent Cloning (LIC)is a fast and easy method for cloning that doesn't use restriction enzymes or DNA ligase. However, for single fragment insertion, we do not believe that the increased resources required are justified when PIPE and SLIC robustly achieve high efficiencies. This allows the exonuclease activity of T4 DNA Polymerase to proceed and generate the complementary overlaps between insert and vector. If that fails or if greater robustness is desired, a restriction enzyme can be used to first cut the template outside of the primer binding sites. 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. After first transforming the DpnI-digested vector PCR product alone we observed a significant number of blue (empty pUC18/Kan) colonies, but no colonies without the PCR (data not shown). How does SLIC compare to other cloning methods? 3 ng). doi: 10.7554/eLife.82843. Open Biol. 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. Supervised the project: AJB. 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 in vivo after transformation into E. coli. FOIA 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. Briefly, the process involves T4 DNA polymerase treatment of linearized vectors in the . Islam MN, Lee KW, Yim HS, Lee SH, Jung HC, Lee JH, Jeong JY. Joined fragments have 4 nicks that are repaired byE.coli during transformation. Unable to load your collection due to an error, Unable to load your delegates due to an error. Bethesda, MD 20894, Web Policies Methods Mol Biol. James R. Krycer, * E-mail: aj.brown@unsw.edu.au (AJB); j.stevenson@unsw.edu.au (JS); j.krycer@garvan.org.au (JRK), Affiliation: The pUC18/Kan vector backbone was amplified with primers pUC18-L30-F and pUC18-L30-R, which are the reverse complement of the common 5-tails of the insert primers. Inserts are usually PCR amplified and vectors are made linear either by restriction enzyme digestion or by PCR. PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US. In 1327, Bishop Gottfried von Osnabrck granted Jews the right to settle in Hamm. Cloning is possible due to the complementary 5 ends of the insert and vector fragments. There is a problem with the plasmid I received. But this didn't yield any positive colonies after DpnI digestion and transformation. Competing interests: The authors have declared that no competing interests exist. Key to SLIC is the power of homologous recombination. Ligation-independent cloning of PCR products (LIC-PCR). The PCR fragment and plasmidare then combined, annealed, and transformed into E. coli. Ligation-independent cloning (LIC) was first developed in the 1990s. & Engineering, Model Gene accession numbers and plasmid vector backbones are listed in Table S2 in File S1. LIC is a reliable cloning method, but it is limited by its sequence constraints. Elledge realized that he could generateimperfect recombination intermediates through PCR and imprecise T4 exonuclease activity, overcoming the requirementforcarefully designed DNA overhangsused in LIC. Figure 3. This should be done in a small volume with no additional water (<5 l). Please sign back in to continue your session. We found that PIPE, SLIC and OEC were also very efficient for site-directed mutagenesis. Thus concentrations of 2550 fmol (1.252.5 nM) were optimal for products >1.5 kb, 50100 fmol (2.55 nM) <1.5 kb, and 100300 fmol (515 nM) <350 bp. Bookshelf -, 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. by PCR, and T4 DNA polymerase in a tube at . 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). SLIC inserts can also be generated by incomplete PCR (iPCR) or mixed PCR. Only one type of dNTP would be present in the reaction mix, limiting the exonuclease activity to the first occurrence of that nucleotide. Coates HW, Capell-Hattam IM, Olzomer EM, Du X, Farrell R, Yang H, Byrne FL, Brown AJ. 2007 Mar;4(3):251-6. A simple method to introduce internal deletions or mutations into any position of a target DNA sequence. Incubate the annealing reaction for 5 minutes at room temperature, then add 1 l of 25 mM EDTA, followed by another 5 minutes at room temperature. This can create multiple distinct overhangs with a single enzyme, and remove the restriction sites from the final product (no "cloning scars"). The methods compared here work either through the generation of complementary single-stranded overhangs for in vivo homologous recombination (PIPE, SLIC) or by generating a nicked plasmid in vitro by overlap extension (OEC). It does not require the use of restriction enzymes and T4 DNA ligase. Biotechniques. Ligation Independent Cloning (LIC) is a technique developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. Careers. 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). Show more Show more 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. official website and that any information you provide is encrypted A practical comparison of ligation-independent cloning techniques The precise assembly of specific DNA sequences is a critical technique in molecular biology. In the second step, the addition of a single dNTP stops the exonuclease reaction. Epub 2023 Jun 8. PMC To directly compare the effectiveness of the different cloning strategies, the reporter vector and different inserts were first amplified and purified, then fed into PIPE, SLIC and OEC with equivalent amounts of DNA using the optimised protocols (as described in the Materials and Methods). 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). Fields, Pathways The annealed but nicked vector product is then repaired during the replication cycle. 2023 Jan 15;11(1):214. doi: 10.3390/microorganisms11010214. These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. , SLIC is a cheap, standardized, and rapid multi-part DNA assembly method - read on to learn how to use it in your research. Addgene is a nonprofit plasmid repository. In SLIC, purified PCR products are treated with T4 DNA polymerase (DNAP) so that the exonuclease activity will increase the proportion of recessed ends. To save your cart and view previous orders, sign in to your NEB account. Epub 2007 Feb 11. A Practical Comparison of Ligation-Independent Cloning Techniques Julian Stevenson, James R. Krycer, Lisa Phan, Andrew J. Two PCR products are used to generate a target gene with a 5 or 3 overhang. 2023 May;13(5):220369. doi: 10.1098/rsob.220369. The ligation independent cloning PCR was initially performed by using megaprimers and empty pHW2000 or pHW2000 containing M gene as a bait plasmid. German chemistry CRO for global pharma, biotech & chemical companies since 1999! SLIC can also be performed using restriction enzyme-cleaved plasmid, minimising primer design at the cost of additional manipulations over PIPE [2], [5], [9]. Figure 4. Have questions about your order, deposit, or a plasmid? Unauthorized use of these marks is strictly prohibited. Nat Commun. If fragments in a multicomponent assembly have 5 or 3 sequence homology to each other, they may be assembled incorrectly. Serial dilutions of transformation mixture were spread onto ampicillin selective plates to allow counting of the number of colony forming units (CFU), adjusted to account for total volume, and rounded to 3 significant figures. However, nicked copies of the vector are often below the limit of detection, yet can still generate significant unwanted background (data not shown). Overall, PIPE achieved cloning efficiencies of 95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. More recently, the technique has evolved to include many useful variations. 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. 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. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. If you don't see your country above, please visit our The .gov means its official. 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. 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. Step 1: Design Your Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested leader sequence fused to your gene of interest, in frame with the start codon or tag sequences (where appropriate). As long as there was enough sequence homology (20-60 bp) to organize the fragments and hold them together. We describe here a method for sequence- and ligation-independent cloning (SLIC). As easy as the technique is, designing primers can be a bit tricky. 2. Performed the experiments: JS JRK LP. This right, however, was limited to wealthy Jews only, as it was depended on a very expensive "Schutzbrief" (letter of protection) by . By continuing to use this site, you agree to the use of cookies. Ligation-independent cloning was our method of choice as it provided a simple and cost-effective tool for producing many constructs of a single target or multiple targets in parallel without the need to select specific restriction enzymes for each gene. Technique selection flowchart for a. The first method of choice is dependent upon the nature of the insert, the availability of existing primers and the level of efficiency required. It is advisable to set up multiple reactions by holding the vector concentration fixed across multiple concentrations of insert, thereby increasing your chances of success. 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. An official website of the United States government. . 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]. ACS Synth Biol. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). 2017 Sep 1;63(3):125-130. doi: 10.2144/000114588. 1. 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. Covering an area of 34,084 square kilometres (13,160 sq mi), it is the fourth-largest German state by size. 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. With more than 18 million inhabitants, it is the most populous state in Germany. However, purification to remove inhibitory buffer components and concentrate the DNA provides better results (Table S12 in File S1 and data not shown). 2007 Mar;4(3):251-6. (2012) One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. . Not only does this system not use site-specific recombination, it also doesnt require a ligation step! Li MZ, Elledge SJ. SLIC is ideal for multicomponent assembly (see figure below), as overlapping sequence homology specifies the order of multiple fragments, and the assembly is scarless. SLIC uses an exonuclease, T4 DNA polymerase, to generate single-stranded DNA overhangs in insert and vector sequences. Larger non-specific products will also compete with the desired insert in PIPE and SLIC, reducing cloning efficiency, which may require gel extraction. This involves designing longer primers for one of the cloning junctions where the vector overhang is complementary to the region within the target insert PCR product, rather than insert primer. 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. Learn more, Please note: Your browser does not fully support some of the features used on Addgene's website. This site needs JavaScript to work properly. HHS Vulnerability Disclosure, Help 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. Traditional cloning makes use of restriction enzymes and ligation of DNA in vitro. This can enable rescue of a failed cloning attempt using a different strategy with a higher efficiency but requiring additional steps and resources (Figure 4 and Table 2). His new method, named sequence- and ligation-independent cloning (SLIC), eliminates many of LICs constraints. Please note: Your browser does not support the features used on Addgene's website. The https:// ensures that you are connecting to the 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) . 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. The effectiveness of OEC was dependent upon megaprimer concentration, with the optimum being inversely proportional to megaprimer size: high concentrations of small insert and vice versa (Table S8 in File S1). government site. A stuffer sequence allows for electrophoretic separation of linearized vector from the reaction mixture, and may provide counter-selection for negative clones as in the example shown here. . Consequently, here we characterise and compare the efficiency, convenience, and utility of three major LIC techniques. Clipboard, Search History, and several other advanced features are temporarily unavailable. Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost. MeSH How do I place an order? Bethesda, MD 20894, Web Policies PIPE cloning is generally simpler and robustly performs well, at the cost of requiring a second primer pair. A 24 bp FLAG epitope insertion (84 bp fragment) was achieved through PIPE or SLIC with primers FLAG-pUC18-F and FLAG-pUC18-R to amplify pUC18/Kan. Another potential issue is sequence similarity. Vector background colonies are generated when the vector plasmid templates are amplified rather than just the insert, or desired vector-backbone product (Figure 2). Learn more, Download our file to copy and paste plasmid data, Learn more about Addgene materials from user-contributed reports describing AAV and antibody experiments, Basic analysis for a user-entered sequence; includes restriction sites and map, Digital collection of empty plasmid backbones from publications and commercially available sources. The pUC18/Kan reporter vector (Figure 1B) was prepared using PIPE cloning. This has proved to be particularly useful for synthetic biology projects requiring assembly of very large DNA fragments. The most effective and convenient methods include polymerase incomplete primer extension (PIPE) cloning [1], sequence and ligation-independent cloning (SLIC) [2], and overlap extension cloning (OEC) [3], [4] (Figure 1). Technique selection flowchart for a new cloning project. 8600 Rockville Pike -, Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. See this image and copyright information in PMC. PIPE or SLIC are optimal for large deletions [1], [10], and extremely effective for substitutions or small insertions like epitope tags (Table 1). and transmitted securely. Technique selection flowchart for a new cloning project. Methods Mol Biol. 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. 1. Thus, positive colonies are white and resistant to both ampicillin and kanamycin (Figure 2C), whilst blue colonies possess pUC18/Kan and Kan-sensitive colonies possess insert-containing plasmids. How do I prepare and deposit my plasmids? Your profile has been mapped to an Institution, please sign back for your profile updates to be completed. This technique allows efficient creation of scarless recombinant plasmids at many, but not all, positions in a vector. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. 5 L fractions were diluted 11 with CutSmart Buffer (to control for buffer and assist DpnI activity), digested for 3 hr at 37C with 20 U of DpnI, and 2 L used for transformation as described above. SLIC uses a brief treatment of purified PCR products with the 35 exonuclease activity of T4 DNA polymerase to generate a higher proportion of recessed ends [2], [5]. Below we use pNIC28-Bsa4 as an example of LIC experimental design. You may not be able to create an account or request plasmids through this website until you upgrade your browser. To make matters even easier, SLIC is also compatible with incompletely synthesized, or iPCR, fragments (right branch of the figure above). The 3-terminal sequence can be removed via the . For very small inserts, using two primer sets to amplify vector and insert would be wasteful and very inefficient for PIPE and SLIC. With molecular cloning scientists can amplify and manipulate genes of interest and then insert them into plasmids for replication and protein expression. We began our optimisation with PIPE. Next Generation Sequencing Library Preparation, DNA Assembly, Cloning and Mutagenesis Kits, Supporting Infectious Disease Research & Development, Some types of sequence modifications not possible. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). The precise assembly of specific DNA sequences is a critical technique in molecular biology. These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. 1994;31:47-55. doi: 10.1385/0-89603-258-2:47. SLIC: a method for sequence- and ligation-independent cloning. To test the ability of the techniques to tolerate the presence of primer-dimer, we attempted to clone a 1.4 kb gene product where the PCR product also included a marked amount of 150 bp primer-dimer or mispriming product. Brown Construction of New Ligation-Independent Cloning Vectors for the Expression and Purification of Recombinant Proteins in Silkworms Using BmNPV Bacmid System PIPE relies on incomplete extension in PCR. This process can occur through one of two pathways: RecA-mediated recombination or RecA-independent single-stranded annealing. When the products are mixed and annealed, 25% of the resulting DNA will have two single-stranded overhangs that can robustly stimulate recombination. Megaprimer and primer-dimer contaminant or 1.4 kb LXR megaprimer alone were gel purified and used for OEC. This allows simple identification of the type of plasmid in each colony insert plasmid, vector plasmid or desired recombinant plasmid as the insert plasmids lack kanamycin resistance, and the vector plasmid template contains undisrupted lacZ. If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. What strain of bacteria does my stab contain? Primer-dimers could be removed by gel extraction or through pre-incubation with T4 polymerase to digest primer-dimer. In contrast, for OEC, most white colonies contained unwanted primer-dimer instead (Figure 3). Disclaimer. SLIC is most often compared to Gibson assembly, another cloning method based on homologous recombination that will be further detailed in an upcoming Plasmids 101 post. 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. Brown, Contributed equally to this work with: 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).
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