Single Tube, High Throughput Cloning of Inverted Repeat Constructs for Double-Stranded RNA Expression
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{"title"=>"Single tube, high throughput cloning of inverted repeat constructs for double-stranded RNA expression", "type"=>"journal", "authors"=>[{"first_name"=>"Brian", "last_name"=>"Hauge", "scopus_author_id"=>"57190387943"}, {"first_name"=>"Christopher", "last_name"=>"Oggero", "scopus_author_id"=>"57199533535"}, {"first_name"=>"Nicole", "last_name"=>"Nguyen", "scopus_author_id"=>"35185500900"}, {"first_name"=>"Changlin", "last_name"=>"Fu", "scopus_author_id"=>"24331266700"}, {"first_name"=>"Fenggao", "last_name"=>"Dong", "scopus_author_id"=>"55244227600"}], "year"=>2009, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-70349682589", "pui"=>"355371443", "doi"=>"10.1371/journal.pone.0007205", "sgr"=>"70349682589", "pmid"=>"19784365"}, "id"=>"88fa53aa-bf80-300d-9e16-bc022ddb0136", "abstract"=>"BACKGROUND: RNA interference (RNAi) has emerged as a powerful tool for the targeted knockout of genes for functional genomics, system biology studies and drug discovery applications. To meet the requirements for high throughput screening in plants we have developed a new method for the rapid assembly of inverted repeat-containing constructs for the in vivo production of dsRNAs.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: The procedure that we describe is based on tagging the sense and antisense fragments with unique single-stranded (ss) tails which are then assembled in a single tube Ligase Independent Cloning (LIC) reaction. Since the assembly reaction is based on the annealing of unique complementary single stranded tails which can only assemble in one orientation, greater than ninety percent of the resultant clones contain the desired insert.\\n\\nCONCLUSION/SIGNIFICANCE: Our single-tube reaction provides a highly efficient method for the assembly of inverted repeat constructs for gene suppression applications. The single tube assembly is directional, highly efficient and readily adapted for high throughput applications.", "link"=>"http://www.mendeley.com/research/single-tube-high-throughput-cloning-inverted-repeat-constructs-doublestranded-rna-expression", "reader_count"=>18, "reader_count_by_academic_status"=>{"Researcher"=>5, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>1, "Other"=>1, "Student > Master"=>3, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Researcher"=>5, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>1, "Other"=>1, "Student > Master"=>3, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Agricultural and Biological Sciences"=>14, "Medicine and Dentistry"=>2, "Arts and Humanities"=>1, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>14}, "Computer Science"=>{"Computer Science"=>1}, "Arts and Humanities"=>{"Arts and Humanities"=>1}}, "reader_count_by_country"=>{"United States"=>1, "China"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/881476"], "description"=>"<p>DNA sequence of the universal loop is shown in panel A Panel B depicts the restriction sites used to linearize the construct for sequence confirmation. The relative position of the sequencing primers is depicted and their sequences are provided.</p>", "links"=>[], "tags"=>["facilitates", "dna", "confirmation"], "article_id"=>551921, "categories"=>["Genetics", "Biotechnology"], "users"=>["Brian Hauge", "Christopher Oggero", "Nicole Nguyen", "Changlin Fu", "Fenggao Dong"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007205.g001", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Universal_loop_facilitates_DNA_sequence_confirmation_of_the_insert_/551921", "title"=>"Universal loop facilitates DNA sequence confirmation of the insert.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 04:23:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/881938"], "description"=>"<p>Gel image showing the annealing of three different Gl1 RNAi constructs The sense, antisense and annealed products are labeled S, AS and Annealed respectively The sense product is larger than the antisense product since it is tethered to the 150 bp universal loop The sizes of the relevant size markers are indicated.</p>", "links"=>[], "tags"=>["visualized", "agarose", "gel"], "article_id"=>552386, "categories"=>["Genetics", "Biotechnology"], "users"=>["Brian Hauge", "Christopher Oggero", "Nicole Nguyen", "Changlin Fu", "Fenggao Dong"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007205.g004", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Annealing_as_visualized_by_agarose_gel_electrophoresis_/552386", "title"=>"Annealing as visualized by agarose gel electrophoresis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 04:26:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/881776"], "description"=>"<p>Diagram depicting UDG mediated ss production of the insert and production of the complementary vector ss tails by restriction digestion (Hpa1) and nicking with the nicking endonuclease (NbBbvCI) Assembly is mediated by efficient annealing of the complementary ss tails.</p>", "links"=>[], "tags"=>["biotechnology", "genetics and genomics", "biotechnology/plant biotechnology", "genetics and genomics/chromosome biology", "genetics and genomics/epigenetics", "genetics and genomics/functional genomics", "genetics and genomics/gene discovery", "genetics and genomics/gene expression", "genetics and genomics/gene function", "genetics and genomics/genomics", "genetics and genomics/plant genetics and gene expression", "genetics and genomics/plant genomes and evolution"], "article_id"=>552212, "categories"=>["Genetics", "Biotechnology"], "users"=>["Brian Hauge", "Christopher Oggero", "Nicole Nguyen", "Changlin Fu", "Fenggao Dong"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007205.g003", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Vector_production_and_insert_annealing_/552212", "title"=>"Vector production and insert annealing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 04:25:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/882046"], "description"=>"<p>Successful cloning is based on the availability of the desired target sequences, so failure to amplify the target sequence is a PCR failure and is not considered a cloning failure. The cloning success rate refers to the number of the desired LIC constructs attained following one round of cloning; this includes incorporation of the uracil containing primers, addition of the loop, and generation of the ss tails and annealing to the vector in the desired orientation.</p>", "links"=>[], "tags"=>["cloning", "inverted", "repeats"], "article_id"=>552500, "categories"=>["Genetics", "Biotechnology"], "users"=>["Brian Hauge", "Christopher Oggero", "Nicole Nguyen", "Changlin Fu", "Fenggao Dong"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007205.t002", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_LIC_cloning_of_inverted_repeats_into_three_different_vectors_/552500", "title"=>"LIC cloning of inverted repeats into three different vectors.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-21 04:27:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/881613"], "description"=>"<p>Four PCR primers are synthesized containing the s5′ and a3′ tags tethered to the forward primer, and the a5′ and s3′ tags tethered to the reverse primer For each target two amplifications are carried out using the a5′/a3′ primer pair and the s5′/s3′ pair to generate the antisense and sense products respectively In the second round of PCR an aliquot of each reaction is amplified using the cognate dU containing primers During the second round of PCR a universal loop is added to the sense product by SOE-PCR The loop is prepared by PCR amplification and a stock is stored at −20°C For the LIC based assembly 5 µl aliquots are mixed with 10 ng of vector, treated with UDG, heated to 65°C and allowed to anneal.</p>", "links"=>[], "tags"=>["lic-based", "palindromic", "rnai"], "article_id"=>552061, "categories"=>["Genetics", "Biotechnology"], "users"=>["Brian Hauge", "Christopher Oggero", "Nicole Nguyen", "Changlin Fu", "Fenggao Dong"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007205.g002", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Strategy_for_LIC_based_assembly_of_palindromic_RNAi_constructs_/552061", "title"=>"Strategy for LIC-based assembly of palindromic RNAi constructs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 04:24:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/882088"], "description"=>"<p>PCR primers used for the assembly of palindromic RNAi constructs The first round PCR sequences correspond to the 5′ tags with are tethered to the gene specific primer sequences The second round PCR primers contain dU residues used to generate ss tails The vector overhang depicts the single stranded regions of the vector which are generated following HpaI and NbBbvCI digestion The left and right ss vector overhangs are complementary to the 3′ tails generated by UDG cleavage of dUa5′ and dUs3′ of their respective PCR products.</p>", "links"=>[], "tags"=>["lic-based"], "article_id"=>552542, "categories"=>["Genetics", "Biotechnology"], "users"=>["Brian Hauge", "Christopher Oggero", "Nicole Nguyen", "Changlin Fu", "Fenggao Dong"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007205.t001", "stats"=>{"downloads"=>1, "page_views"=>21, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primers_for_LIC_based_assembly_/552542", "title"=>"Primers for LIC-based assembly.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-21 04:27:14"}

PMC Usage Stats | Further Information

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