Sticky-end PCR: new method for subcloning.

Abstract

BioTechniquesVol. 25, No. 2 BenchmarksOpen AccessSticky-End PCR: New Method for SubcloningGang ZengGang Zeng*Address correspondence to Dr. Gang Zeng, Surgery Branch, Room 4B50, Bldg 10, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Internet: E-mail Address: gang_zeng@nih.govGeorgetown University Medical Center, Washington, DC, USASearch for more papers by this authorPublished Online:15 Aug 2018https://doi.org/10.2144/98252bm05AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited BySynergic action of an inserted carbohydrate-binding module in a glycoside hydrolase family 5 endoglucanase20 April 2022 | Acta Crystallographica Section D Structural Biology, Vol. 78, No. 5A novel DNA primase-helicase pair encoded by SCCmec elements18 September 2020 | eLife, Vol. 9Bio-Fenton reaction involved in the cleavage of the ethoxylate chain of nonionic surfactants by dihydrolipoamide dehydrogenase from Pseudomonas nitroreducens TX12 May 2019 | Scientific Reports, Vol. 9, No. 1Protein-coated dsDNA nanostars with high structural rigidity and high enzymatic and thermal stability1 January 2019 | Nanoscale, Vol. 11, No. 40Re-investigating substrate specificity of dihydroflavonol 4-reductase with respect to the B-ring hydroxylation pattern of substratesActa Horticulturae, No. 1242Crystal Structure of a Putative Modulator of Gyrase (TldE) from Thermococcus kodakarensis19 February 2019 | Crystals, Vol. 9, No. 2RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula)9 June 2018 | Planta, Vol. 249, No. 1Association of a functional TNF variant with Plasmodium falciparum parasitaemia in a congolese population13 July 2017 | Genes & Immunity, Vol. 18, No. 3Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation17 July 2017 | Journal of the American Chemical Society, Vol. 139, No. 29Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia3 March 2017 | Plant Molecular Biology, Vol. 93, No. 6Synthetic Biology Standards and Methods of DNA Assembly7 April 2016Molecular Characterization of Ethylene Response Sensor 1 (BoERS1) in Bambusa oldhamii21 August 2015 | Plant Molecular Biology Reporter, Vol. 34, No. 2Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues9 August 2015 | Plant Molecular Biology, Vol. 89, No. 1-2Deciphering the Roles of Multicomponent Recognition Signals by the AAA + Unfoldase ClpXJournal of Molecular Biology, Vol. 427, No. 18A Derivative of the Thiopeptide GE2270A Highly Selective against Propionibacterium acnes18 May 2015 | Antimicrobial Agents and Chemotherapy, Vol. 59, No. 8Characterization and Inhibition of the Main Protease of Severe Acute Respiratory Syndrome Coronavirus25 February 2015 | ChemBioEng Reviews, Vol. 2, No. 2Interleukin-4 up-regulation of epidermal interleukin-19 expression in keratinocytes involves the binding of signal transducer and activator of transcription 6 (Stat6) to the imperfect Stat6 sites10 November 2014 | Immunology, Vol. 143, No. 4Isolation of Dihydroflavonol 4-Reductase cDNA Clones from Angelonia x angustifolia and Heterologous Expression as GST Fusion Protein in Escherichia coli19 September 2014 | PLoS ONE, Vol. 9, No. 9Transcriptome profiling, and cloning and characterization of the main monoterpene synthases of Coriandrum sativum L.Phytochemistry, Vol. 102Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo14 April 2014 | PLoS ONE, Vol. 9, No. 4Development of a genomic engineering tool in Saccharomyces cerevisiaeJournal of the Taiwan Institute of Chemical Engineers, Vol. 45, No. 1Schistosoma mansoni tetraspanning orphan receptor (SmTOR): a new vaccine candidate against schistosomiasis1 November 2012 | Clinical and Experimental Immunology, Vol. 170, No. 3Rad51 Is an Accessory Factor for Dmc1-Mediated Joint Molecule Formation During MeiosisScience, Vol. 337, No. 6099Cloning, functional characterization and genomic organization of 1,8-cineole synthases from Lavandula17 May 2012 | Plant Molecular Biology, Vol. 79, No. 4-5HDL-like discs for assaying membrane proteins in drug discoveryBiophysical Chemistry, Vol. 165-166The Metal Core Structures in the Recombinant Escherichia coli Transcriptional Factor SoxR20 January 2012 | Chemistry - A European Journal, Vol. 18, No. 9Substrate specificity and contribution of the glycosyltransferase UGT71A15 to phloridzin biosynthesis28 December 2011 | Trees, Vol. 26, No. 1Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Piceaspp.)7 March 2011 | BMC Plant Biology, Vol. 11, No. 1A kinetic comparison of asparagine synthetase isozymes from higher plantsPlant Physiology and Biochemistry, Vol. 49, No. 3Production of heparanase constructs suitable for nuclear magnetic resonance and drug discovery studies29 September 2010 | Biopolymers, Vol. 95, No. 2Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera) Terpene Synthase Gene Family Based on Genome Assembly, FLcDNA Cloning, and Enzyme Assays21 October 2010 | BMC Plant Biology, Vol. 10, No. 1Characterization of plant p23-like proteins for their co-chaperone activities28 March 2010 | Cell Stress and Chaperones, Vol. 15, No. 5The Structure of Ca2+ Sensor Case16 Reveals the Mechanism of Reaction to Low Ca2+ Concentrations30 August 2010 | Sensors, Vol. 10, No. 9Temperature-sensitive Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases Is Mediated by the Endoplasmic Reticulum-associated Degradation PathwayJournal of Biological Chemistry, Vol. 285, No. 28The X-ray Crystal Structure of the First RNA Recognition Motif and Site-Directed Mutagenesis Suggest a Possible HuR Redox Sensing MechanismJournal of Molecular Biology, Vol. 397, No. 5Identification and Functional Characterization of Monofunctional ent -Copalyl Diphosphate and ent -Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms31 December 2009 | Plant Physiology, Vol. 152, No. 3Chimeric adenoviral vectors incorporating a fiber of human adenovirus 3 efficiently mediate gene transfer into prostate cancer cells9 November 2009 | The Prostate, Vol. 70, No. 4Point Mutations (Q19P and N23K) Increase the Operational Solubility of a 2α- O -Benzoyltransferase that Conveys Various Acyl Groups from CoA to a Taxane Acceptor28 January 2010 | Journal of Natural Products, Vol. 73, No. 2Engineering a novel endopeptidase based on SARS 3CLproChih-Jung Kuo, Yan-Ping Shih, Daphne Kan & Po-Huang Liang25 April 2018 | BioTechniques, Vol. 47, No. 6Isolation and Characterization of AaWRKY1, an Artemisia annua Transcription Factor that Regulates the Amorpha-4,11-diene Synthase Gene, a Key Gene of Artemisinin Biosynthesis24 September 2009 | Plant and Cell Physiology, Vol. 50, No. 12Structural Insight into Translesion Synthesis by DNA Pol IICell, Vol. 139, No. 7The Drosophila G protein-coupled receptor, Methuselah, exhibits a promiscuous response to peptides11 August 2009 | Protein Science, Vol. 18, No. 11Delivering multiple gene products in the brain from a single adeno-associated virus vector3 September 2009 | Gene Therapy, Vol. 16, No. 11Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: Functional divergence of the dilysine ER retrieval motif in plant cellsPlant Physiology and Biochemistry, Vol. 47, No. 10Preparation of long sticky ends for universal ligation-independent cloning: Sequential T4 DNA polymerase treatmentsJournal of Bioscience and Bioengineering, Vol. 107, No. 6Structural Adaptations that Modulate Monosaccharide, Disaccharide, and Trisaccharide Specificities in Periplasmic Maltose-Binding ProteinsJournal of Molecular Biology, Vol. 389, No. 1Sticky PCR: A PCR-based protocol for targeted protein engineeringBiotechnology Journal, Vol. 4, No. 4HHP1 is involved in osmotic stress sensitivity in Arabidopsis13 March 2009 | Journal of Experimental Botany, Vol. 60, No. 6Parallel gene cloning and protein production in multiple expression systemsBiotechnology Progress, Vol. 328The Taxol Pathway 10- O -Acetyltransferase Shows Regioselective Promiscuity with the Oxetane Hydroxyl of 4-Deacetyltaxanes14 November 2008 | Journal of the American Chemical Society, Vol. 130, No. 50The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog28 March 2008 | BMC Structural Biology, Vol. 8, No. 1Ligand-induced conformational changes in a thermophilic ribose-binding protein19 November 2008 | BMC Structural Biology, Vol. 8, No. 1Structural Analysis of a Periplasmic Binding Protein in the Tripartite ATP-independent Transporter Family Reveals a Tetrameric Assembly That May Have a Role in Ligand TransportJournal of Biological Chemistry, Vol. 283, No. 47Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood, Santalum album L.Archives of Biochemistry and Biophysics, Vol. 477, No. 1Crystal structure of Tflp: A ferredoxin-like metallo-β-lactamase superfamily protein from Thermoanaerobacter tengcongensis22 April 2008 | Proteins: Structure, Function, and Bioinformatics, Vol. 72, No. 1Development of a novel Gateway-based vector system for efficient, multiparallel protein expression in Escherichia coliProtein Expression and Purification, Vol. 59, No. 2Functional plasticity of paralogous diterpene synthases involved in conifer defense15 January 2008 | Proceedings of the National Academy of Sciences, Vol. 105, No. 3Testing of a heterologous, wound- and insect-inducible promoter for functional genomics studies in conifer defense2 August 2007 | Plant Cell Reports, Vol. 26, No. 12Unusual Mechanism for an Aminomutase Rearrangement: Retention of Configuration at the Migration Termini4 August 2007 | Biochemistry, Vol. 46, No. 34Expression of zinc finger transcription factor Bcl11A/Evi9/CTIP1 in rat brainJournal of Neuroscience Research, Vol. 85, No. 8Enzyme Free Cloning for high throughput gene cloning and expression13 February 2007 | Journal of Structural and Functional Genomics, Vol. 7, No. 3-4The Crystal Structure of a Thermophilic Glucose Binding Protein Reveals Adaptations that Interconvert Mono and Di-saccharide Binding SitesJournal of Molecular Biology, Vol. 362, No. 2Evaluation of two novel tag-based labelling technologies for site-specific modification of proteinsInternational Journal of Biological Macromolecules, Vol. 39, No. 1-3Pinus taeda phenylpropenal double-bond reductase: Purification, cDNA cloning, heterologous expression in Escherichia coli, and subcellular localization in P. taedaPhytochemistry, Vol. 67, No. 16Substrate specificity of Arabidopsis 3-ketoacyl-CoA synthasesBiochemical and Biophysical Research Communications, Vol. 346, No. 2A Conserved Dimer and Global Conformational Changes in the Structure of apo-PknE Ser/Thr Protein Kinase from Mycobacterium tuberculosisJournal of Molecular Biology, Vol. 360, No. 2Adeno-associated Virus-Mediated Expression and Constitutive Secretion of Galanin Suppresses Limbic Seizure Activity in VivoMolecular Therapy, Vol. 14, No. 1Conformation-specific affinity purification of proteins using engineered binding proteins: Application to the estrogen receptorProtein Expression and Purification, Vol. 47, No. 2Preparation of Sticky-End PCR Products and Ligation into Expression Vectors for High-Throughput Screening of Soluble Recombinant Proteins1 January 2006 | Cold Spring Harbor Protocols, Vol. 2006, No. 1A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic ArabidopsisThe Plant Journal, Vol. 45, No. 5Rapid and easy thermodynamic optimization of the 5′-end of mRNA dramatically increases the level of wild type protein expression in Escherichia coliProtein Expression and Purification, Vol. 45, No. 2Regulation of PLCβ Isoforms by RacThe cAMP Pathway Regulates Both Transcription and Activity of the Paired Homeobox Transcription Factor Phox2a Required for Development of Neural Crest-derived and Central Nervous System-derived Catecholaminergic NeuronsJournal of Biological Chemistry, Vol. 280, No. 49Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2Analytical Biochemistry, Vol. 343, No. 2Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cellsProtein Expression and Purification, Vol. 42, No. 1Complex mosaicism is a novel approach to infectivity enhancement of adenovirus type 5-based vectors11 February 2005 | Cancer Gene Therapy, Vol. 12, No. 5Monoterpene Metabolism. Cloning, Expression, and Characterization of (−)-Isopiperitenol/(−)-Carveol Dehydrogenase of Peppermint and Spearmint10 March 2005 | Plant Physiology, Vol. 137, No. 3Monoterpene Metabolism. Cloning, Expression, and Characterization of Menthone Reductases from Peppermint10 March 2005 | Plant Physiology, Vol. 137, No. 3A Baculovirus Expression Vector System for Simultaneous Protein Expression in Insect and Mammalian Cells5 September 2008 | Biotechnology Progress, Vol. 21, No. 3Identification of Tbr-1/CASK complex target genes in neurons2 December 2004 | Journal of Neurochemistry, Vol. 91, No. 6Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol BiosynthesisJournal of Biological Chemistry, Vol. 279, No. 52An Alternate Conformation and a Third Metal in PstP/Ppp, the M. tuberculosis PP2C-Family Ser/Thr Protein PhosphataseStructure, Vol. 12, No. 11A cryptic lysis gene near the start of the Qβ replicase gene in the +1 frame6 October 2004 | Genes to Cells, Vol. 9, No. 10Functional Characterization of Nine Norway Spruce TPS Genes and Evolution of Gymnosperm Terpene Synthases of the TPS-d Subfamily 23 August 2004 | Plant Physiology, Vol. 135, No. 4Altering product outcome in Abies grandis (−)-limonene synthase and (−)-limonene/(−)-α-pinene synthase by domain swapping and directed mutagenesisArchives of Biochemistry and Biophysics, Vol. 425, No. 1Sensor Domain of the Mycobacterium tuberculosis Receptor Ser/Thr Protein Kinase, PknD, forms a Highly Symmetric β PropellerJournal of Molecular Biology, Vol. 339, No. 2Biochemical and Molecular Characterization of ACH2, an Acyl-CoA Thioesterase from Arabidopsis thalianaJournal of Biological Chemistry, Vol. 279, No. 9Genetically Targeted Adenovirus Vector Directed to CD40-Expressing CellsJournal of Virology, Vol. 77, No. 21Monoterpene double-bond reductases of the (−)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (−)-isopiperitenone reductase and (+)-pulegone reductase of peppermintArchives of Biochemistry and Biophysics, Vol. 418, No. 1Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion13 July 2003 | Nature Medicine, Vol. 9, No. 8A Rubredoxin based system for screening of protein expression conditions and on-line monitoring of the purification processProtein Expression and Purification, Vol. 28, No. 2Structure and allosteric regulation of the αXβ2 integrin I domain28 January 2003 | Proceedings of the National Academy of Sciences, Vol. 100, No. 4The Maize Gene terpene synthase 1 Encodes a Sesquiterpene Synthase Catalyzing the Formation of ( E )-β-Farnesene, ( E )-Nerolidol, and ( E , E )-Farnesol after Herbivore Damage6 December 2002 | Plant Physiology, Vol. 130, No. 4Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure SensitivityMolecular Therapy, Vol. 6, No. 4Molecular cloning and characterization of a new linalool synthaseArchives of Biochemistry and Biophysics, Vol. 405, No. 1Modulation of Adenovirus Vector Tropism via Incorporation of Polypeptide Ligands into the Fiber ProteinJournal of Virology, Vol. 76, No. 17Arabidopsis Contains Nine Long-Chain Acyl-Coenzyme A Synthetase Genes That Participate in Fatty Acid and Glycerolipid Metabolism1 August 2002 | Plant Physiology, Vol. 129, No. 4The final acylation step in Taxol biosynthesis: Cloning of the taxoid C13-side-chain N -benzoyltransferase from Taxus27 June 2002 | Proceedings of the National Academy of Sciences, Vol. 99, No. 14High-throughput screening of soluble recombinant proteins13 April 2009 | Protein Science, Vol. 11, No. 7Alteration of product formation by directed mutagenesis and truncation of the multiple-product sesquiterpene synthases δ-selinene synthase and γ-humulene synthaseArchives of Biochemistry and Biophysics, Vol. 402, No. 1Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay30 April 2002 | European Journal of Biochemistry, Vol. 269, No. 10Gene expression of 5-epi-aristolochene synthase and formation of capsidiol in roots of Nicotiana attenuata and N. sylvestrisPhytochemistry, Vol. 60, No. 2Chemical denaturation and elevated folding temperatures are required for wild-type activity and stability of recombinant Methanococcus jannaschii 20S proteasome13 April 2009 | Protein Science, Vol. 10, No. 9Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-Å resolution12 December 2000 | Proceedings of the National Academy of Sciences, Vol. 97, No. 26Identification of Amino Acid Residues in CD81 Critical for Interaction with Hepatitis C Virus Envelope Glycoprotein E2Journal of Virology, Vol. 74, No. 8Terpenoid Secondary Metabolism in Arabidopsis thaliana: cDNA Cloning, Characterization, and Functional Expression of a Myrcene/(E)-β-Ocimene SynthaseArchives of Biochemistry and Biophysics, Vol. 375, No. 2Limited T4 Exonuclease Activity and Partial Fill-in Expand Insertion Site Options for PCR SubcloningNicole Green, Son Vu, Shadi Farahmand & Sandra B. Sharp22 August 2018 | BioTechniques, Vol. 27, No. 5Biosynthesis of Isoprenoids in Escherichia coli : Stereochemistry of the Reaction Catalyzed by Farnesyl Diphosphate Synthase11 September 1999 | Organic Letters, Vol. 1, No. 7 Vol. 25, No. 2 Follow us on social media for the latest updates Metrics Downloaded 1,634 times History Published online 15 August 2018 Published in print August 1998 Information© 2018 Author(s)PDF download