A Practical Comparison of Ligation-Independent Cloning Techniques
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{"title"=>"A practical comparison of ligation-independent cloning techniques", "type"=>"journal", "authors"=>[{"first_name"=>"Julian", "last_name"=>"Stevenson", "scopus_author_id"=>"26642264800"}, {"first_name"=>"James R.", "last_name"=>"Krycer", "scopus_author_id"=>"23501986000"}, {"first_name"=>"Lisa", "last_name"=>"Phan", "scopus_author_id"=>"55340075100"}, {"first_name"=>"Andrew J.", "last_name"=>"Brown", "scopus_author_id"=>"35516561200"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84893498225", "doi"=>"10.1371/journal.pone.0083888", "pui"=>"372292914", "pmid"=>"24376768", "scopus"=>"2-s2.0-84893498225", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "arxiv"=>"arXiv:1011.1669v3"}, "id"=>"fede7af6-c024-3a23-b70b-c911c39522d3", "abstract"=>"The precise assembly of specific DNA sequences is a critical technique in molecular biology. 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. 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). 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. 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. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of approximately 95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. 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. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.", "link"=>"http://www.mendeley.com/research/practical-comparison-ligationindependent-cloning-techniques", "reader_count"=>208, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>10, "Researcher"=>51, "Student > Doctoral Student"=>7, "Student > Ph. D. Student"=>52, "Student > Postgraduate"=>9, "Student > Master"=>25, "Other"=>7, "Student > Bachelor"=>40, "Lecturer"=>3, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>10, "Researcher"=>51, "Student > Doctoral Student"=>7, "Student > Ph. D. Student"=>52, "Student > Postgraduate"=>9, "Student > Master"=>25, "Other"=>7, "Student > Bachelor"=>40, "Lecturer"=>3, "Professor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>5, "Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>56, "Agricultural and Biological Sciences"=>126, "Medicine and Dentistry"=>2, "Veterinary Science and Veterinary Medicine"=>2, "Chemical Engineering"=>3, "Physics and Astronomy"=>3, "Chemistry"=>4, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>5}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Chemistry"=>{"Chemistry"=>4}, "Physics and Astronomy"=>{"Physics and Astronomy"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>126}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>56}, "Unspecified"=>{"Unspecified"=>4}, "Chemical Engineering"=>{"Chemical Engineering"=>3}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>2}}, "reader_count_by_country"=>{"Belgium"=>3, "United States"=>4, "Uruguay"=>1, "Japan"=>2, "Finland"=>1, "Brazil"=>1, "Denmark"=>1, "Mexico"=>1, "Portugal"=>1, "Germany"=>2, "Estonia"=>1}, "group_count"=>7}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1327151"], "description"=>"<p>(A) In PIPE, incomplete extension during PCR generates 3′-recessed ends. In SLIC, purified PCR products are treated with T4 DNA polymerase (DNAP) so that the exonuclease activity will increase the proportion of recessed ends. 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 <i>in vivo</i> after transformation into <i>E. coli.</i> In OEC, the insert PCR product acts a megaprimer to generate a nicked plasmid by overlap extension <i>in vitro</i> in a second round of amplification. The nicks are also repaired <i>in vivo</i>. For all techniques, a DpnI digestion step is included to remove plasmid template (not shown). (B) Design of the reporter vector, encoding resistance for ampicillin and kanamycin, and the alpha-fragment of beta-galactosidase. (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.</p>", "links"=>[], "tags"=>["Biochemistry", "biotechnology", "Applied microbiology", "Environmental biotechnology", "genetics", "Gene splicing", "Molecular genetics", "microbiology", "Molecular cell biology", "Nucleic acids", "dna", "Synthetic Biology", "polymerase", "incomplete", "primer", "ligation-independent", "cloning", "overlap"], "article_id"=>885552, "categories"=>["Biological Sciences"], "users"=>["Julian Stevenson", "James R. Krycer", "Lisa Phan", "Andrew J. Brown"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083888.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Principles_of_polymerase_incomplete_primer_extension_PIPE_cloning_sequence_and_ligation_independent_cloning_SLIC_and_overlap_extension_cloning_OEC_/885552", "title"=>"Principles of polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC) and overlap extension cloning (OEC).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:49:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327153"], "description"=>"<p>Unwanted copies of the original vector plasmid template can be generated by overlap extension in the process of obtaining vector-backbone PCR product (A). This can be prevented by reducing the template concentration (B), cutting the plasmid template first or cloning into a vector with an existing large insert (C). See main text for details.</p>", "links"=>[], "tags"=>["Biochemistry", "biotechnology", "Applied microbiology", "Environmental biotechnology", "genetics", "Gene splicing", "Molecular genetics", "microbiology", "Molecular cell biology", "Nucleic acids", "dna", "Synthetic Biology", "nicked", "vector", "plasmid", "cloning"], "article_id"=>885554, "categories"=>["Biological Sciences"], "users"=>["Julian Stevenson", "James R. Krycer", "Lisa Phan", "Andrew J. Brown"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083888.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_nicked_vector_plasmid_can_reduce_PIPE_cloning_efficiency_/885554", "title"=>"Generation of nicked vector plasmid can reduce PIPE cloning efficiency.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:49:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327155"], "description"=>"<p>Megaprimer and primer-dimer contaminant or 1.4 kb LXR megaprimer alone were gel purified and used for OEC. Ten white colonies for each were screened by PCR across the cloning junctions.</p>", "links"=>[], "tags"=>["Biochemistry", "biotechnology", "Applied microbiology", "Environmental biotechnology", "genetics", "Gene splicing", "Molecular genetics", "microbiology", "Molecular cell biology", "Nucleic acids", "dna", "Synthetic Biology"], "article_id"=>885556, "categories"=>["Biological Sciences"], "users"=>["Julian Stevenson", "James R. Krycer", "Lisa Phan", "Andrew J. Brown"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083888.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_OEC_does_not_tolerate_primer_dimers_/885556", "title"=>"OEC does not tolerate primer-dimers.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:49:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327156"], "description"=>"<p>The number of fragments, their size, primer availability and the presence of primer-dimers will determine the optimal cloning strategy. See main text for details.</p>", "links"=>[], "tags"=>["Biochemistry", "biotechnology", "Applied microbiology", "Environmental biotechnology", "genetics", "Gene splicing", "Molecular genetics", "microbiology", "Molecular cell biology", "Nucleic acids", "dna", "Synthetic Biology", "flowchart", "cloning"], "article_id"=>885557, "categories"=>["Biological Sciences"], "users"=>["Julian Stevenson", "James R. Krycer", "Lisa Phan", "Andrew J. Brown"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083888.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Technique_selection_flowchart_for_a_new_cloning_project_/885557", "title"=>"Technique selection flowchart for a new cloning project.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 03:49:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327157"], "description"=>"<p>See main text for details.</p><p><sup>a</sup> Only 1 primer pair is required for SLIC if cleaved plasmid vector is used.</p><p><sup>b</sup> Optional for small megaprimers (∼500 bp).</p>", "links"=>[], "tags"=>["Biochemistry", "biotechnology", "Applied microbiology", "Environmental biotechnology", "genetics", "Gene splicing", "Molecular genetics", "microbiology", "Molecular cell biology", "Nucleic acids", "dna", "Synthetic Biology"], "article_id"=>885558, "categories"=>["Biological Sciences"], "users"=>["Julian Stevenson", "James R. Krycer", "Lisa Phan", "Andrew J. Brown"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083888.t002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Summary_of_effectiveness_and_resource_use_/885558", "title"=>"Summary of effectiveness and resource use.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-12-23 03:49:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327158"], "description"=>"<p>See main text for details.</p><p><sup>a</sup> Single representative experiments are shown for FLAG (85 bp), Gluc (350 bp), Insig-1 (1.4 kb), and SCAP (4.3 kb).</p><p><sup>b</sup> The increase in colonies relative to PIPE is shown.</p>", "links"=>[], "tags"=>["Biochemistry", "biotechnology", "Applied microbiology", "Environmental biotechnology", "genetics", "Gene splicing", "Molecular genetics", "microbiology", "Molecular cell biology", "Nucleic acids", "dna", "Synthetic Biology", "slic", "oec"], "article_id"=>885559, "categories"=>["Biological Sciences"], "users"=>["Julian Stevenson", "James R. Krycer", "Lisa Phan", "Andrew J. Brown"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083888.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Direct_comparison_of_PIPE_SLIC_and_OEC_for_various_insert_sizes_/885559", "title"=>"Direct comparison of PIPE, SLIC and OEC for various insert sizes.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-12-23 03:49:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/1327162", "https://ndownloader.figshare.com/files/1327163", "https://ndownloader.figshare.com/files/1327164"], "description"=>"<div><p>The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA <i>in vitro</i>, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. 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). 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. 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. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. 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. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.</p></div>", "links"=>[], "tags"=>["Biochemistry", "biotechnology", "Applied microbiology", "Environmental biotechnology", "genetics", "Gene splicing", "Molecular genetics", "microbiology", "Molecular cell biology", "Nucleic acids", "dna", "Synthetic Biology", "ligation-independent", "cloning"], "article_id"=>885563, "categories"=>["Biological Sciences"], "users"=>["Julian Stevenson", "James R. Krycer", "Lisa Phan", "Andrew J. Brown"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083888.s001", "https://dx.doi.org/10.1371/journal.pone.0083888.s002", "https://dx.doi.org/10.1371/journal.pone.0083888.s003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_Practical_Comparison_of_Ligation_Independent_Cloning_Techniques_/885563", "title"=>"A Practical Comparison of Ligation-Independent Cloning Techniques", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-12-23 03:49:58"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"62", "full-text"=>"65", "pdf"=>"10", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"5", "cited-by"=>"0", "year"=>"2020", "month"=>"2"}
  • {"unique-ip"=>"71", "full-text"=>"74", "pdf"=>"16", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2020", "month"=>"3"}
  • {"unique-ip"=>"71", "full-text"=>"87", "pdf"=>"16", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2020", "month"=>"4"}

Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[266, 468, 593, 703, 804, 903, 993, 1084, 1171, 1256, 1339, 1422, 1492]}, {"subject_area"=>"/Biology and life sciences/Biotechnology", "average_usage"=>[259, 472, 622, 759, 885, 1009, 1123, 1232, 1335, 1437, 1533, 1627, 1698]}]}
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