Solid-phase cloning to create sublibraries suitable for DNA sequencing.


:A solid-phase method is described to create subclones, suitable for DNA sequencing, from lambda or cosmid libraries. The purified target DNA is sonicated and two linkers, with one oligonucleotide biotinylated in the 5'-end, are ligated to the ends of the fragments produced by sonication. After size separation, the fragments are immobilised onto a solid support and the non-biotinylated strand of each immobilised fragment is eluted. In this way, a library of single-stranded fragments is obtained. All fragments contain 'universal' flanking sequences of 22 bases introduced by the linker ligation. These flanking sequences can subsequently be used for solid-phase cloning into a single-stranded vector containing the complementary sequences. Thus, cloning can be achieved without the use of ligase or restriction enzymes. The resulting subclones are used for direct solid-phase sequencing and the immobilised strand can be used to selectively remove homologous DNA from the library of single-stranded fragments. Thus, a sublibrary of non-sequenced fragments can be created. Here, we show that a library of clones, suitable for direct solid-phase sequencing, can be obtained starting with lambda DNA. The efficiency of selective hybridisation of homologous and non-homologous fragments was investigated. The possibility of using this approach for automated cloning strategies for large-scale genomic and cDNA sequencing is discussed.


J Biotechnol


Journal of biotechnology


Hultman T,Uhlén M




Has Abstract


1994-06-30 00:00:00














  • Metabolic flux analysis gives an insight on verapamil induced changes in central metabolism of HL-1 cells.

    abstract::Verapamil has been shown to inhibit glucose transport in several cell types. However, the consequences of this inhibition on central metabolism are not well known. In this study we focused on verapamil induced changes in metabolic fluxes in a murine atrial cell line (HL-1 cells). These cells were adapted to serum free...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Strigun A,Noor F,Pironti A,Niklas J,Yang TH,Heinzle E

    更新日期:2011-09-20 00:00:00

  • Morphologically-structured models of growing budding yeast populations.

    abstract::It has been well recognized that many key aspects of cell cycle regulation are encoded into the size distributions of growing budding yeast populations due to the tight coupling between cell growth and cell division present in this organism. Several attempts have been made to model the cell size distribution of growin...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Hatzis C,Porro D

    更新日期:2006-07-13 00:00:00

  • Production of the sesquiterpene (+)-valencene by metabolically engineered Corynebacterium glutamicum.

    abstract::The sesquiterpene (+)-valencene is an aroma compound of citrus fruits and is used to flavor foods and drinks. Biosynthesis of (+)-valencene starts from farnesyl pyrophosphate, an intermediate of carotenoid biosynthesis. Corynebacterium glutamicum, the workhorse of the million-ton scale amino acid industry, is naturall...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Frohwitter J,Heider SA,Peters-Wendisch P,Beekwilder J,Wendisch VF

    更新日期:2014-12-10 00:00:00

  • Kinetic characterization of the photosynthetic reaction centres in microalgae by means of fluorescence methodology.

    abstract::The kinetic characterization of the photosynthetic activity in autotrophic microalgae plays a key role in the design of optimized photobioreactors. This paper presents a procedure to assess kinetic parameters of a three-state photosynthetic reaction centres model. Four kinetic parameters of the model were assessed by ...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Gargano I,Olivieri G,Spasiano D,Andreozzi R,Pollio A,Marotta R,D'Ambrosio N,Marzocchella A

    更新日期:2015-10-20 00:00:00

  • Expression and trafficking of fluorescent viral membrane proteins in baculovirus-transduced BHK cells.

    abstract::Baculovirus vectors show promise as a novel tool for gene delivery into mammalian cells and gene transfer with wild-type baculovirus has been demonstrated both in vitro and in vivo. To study expression and intracellular trafficking of foreign viral membrane proteins in baculovirus-transduced mammalian cells, the envel...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Ojala K,Tikka PJ,Kautto L,Käpylä P,Marjomäki V,Oker-Blom C

    更新日期:2004-10-19 00:00:00

  • Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines.

    abstract::One potential reason for the enhancement of immune responses to DNA vaccines following electroporation is increased gene expression. However, the inflammatory response and accompanying cellular infiltration stimulated by electroporation may also be essential for enhancing immune responses to DNA vaccines. These parame...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Babiuk S,Baca-Estrada ME,Foldvari M,Middleton DM,Rabussay D,Widera G,Babiuk LA

    更新日期:2004-05-13 00:00:00

  • Genome-wide analysis of the L-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation.

    abstract::The genome sequence of Corynebacterium glutamicum, a gram-positive soil bacterium widely used as an amino acid producer, was analyzed by a similarity-based approach to elucidate the pathway for the biosynthesis of L-methionine. The functions of candidate ORFs were derived by gene deletion and, if necessary, by homolog...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Rückert C,Pühler A,Kalinowski J

    更新日期:2003-09-04 00:00:00

  • Development of biodegradable scaffolds based on patient-specific arterial configuration.

    abstract::Biodegradable scaffolds are of great value in tissue engineering. We have developed a method for fabricating patient-specific vascular scaffolds from a biocompatible and biodegradable polymer, poly(L-lactide-co-epsilon-caprolactone). This method's usefulness is due to flexibility in the choice of materials and vascula...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Uchida T,Ikeda S,Oura H,Tada M,Nakano T,Fukuda T,Matsuda T,Negoro M,Arai F

    更新日期:2008-01-20 00:00:00

  • Complete genome sequence of Thialkalivibrio versutus D301 isolated from Soda Lake in northern China, a typical strain with great ability to oxidize sulfide.

    abstract::Thioalkalivibrio versutus D301 isolated from Soda Lake is a haloalkaliphilic and obligated chemolithoautotrophic Gram-negative bacterium. The strain has a good adaption to hyperhaline and highly alkaline environment and a powerful sulfur-oxidizing ability. Here, we present the complete genome sequence of T. versutus D...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Mu T,Zhou J,Yang M,Xing J

    更新日期:2016-06-10 00:00:00

  • Gene prediction and gene classes in Arabidopsis thaliana.

    abstract::Gene prediction methods for eukaryotic genomes still are not fully satisfying. One way to improve gene prediction accuracy, proven to be relevant for prokaryotes, is to consider more than one model of genes. Thus, we used our classification of Arabidopsis thaliana genes in two classes (CU(1) and CU(2)), previously del...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Mathé C,Déhais P,Pavy N,Rombauts S,Van Montagu M,Rouzé P

    更新日期:2000-03-31 00:00:00

  • Production of targeted poly(3-hydroxyalkanoates) copolymers by glycogen accumulating organisms using acetate as sole carbon source.

    abstract::One of the main limitations in bacterial polyhydroxyalkanoate (PHA) production with mixed cultures is the fact that primarily polyhydroxybutyrate (PHB) homopolymers are generated from acetate as the main carbon source, which is brittle and quite fragile. The incorporation of different 3-hydroxyalkanoate (HA) component...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Dai Y,Yuan Z,Jack K,Keller J

    更新日期:2007-05-01 00:00:00

  • Co-expression of disulfide oxidoreductases DsbA/DsbC markedly enhanced soluble and functional expression of reteplase in Escherichia coli.

    abstract::Reteplase is the third generation of thrombolytic medicine and has many advantages over commercial t-PA. However, over-expressing recombinant reteplase in E. coli always accumulates as inclusion bodies due to nine pairs of disulfide bonds formation that is the main obstacle for correct folding. In this paper, in order...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Zhuo XF,Zhang YY,Guan YX,Yao SJ

    更新日期:2014-12-20 00:00:00

  • Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens.

    abstract::The production of the blue pigment indigoidine has been achieved in the entomopathogenic bacterium Photorhabdus luminescens by a promoter exchange and in Escherichia coli following heterologous expression of the biosynthesis gene indC. Moreover, genes involved in the regulation of this previously "silent" biosynthesis...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Brachmann AO,Kirchner F,Kegler C,Kinski SC,Schmitt I,Bode HB

    更新日期:2012-01-01 00:00:00

  • Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    abstract::Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Pol...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Suzuki T,Ezure T,Ando E,Nishimura O,Utsumi T,Tsunasawa S

    更新日期:2010-01-01 00:00:00

  • Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

    abstract::The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). T...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Schenk JA,Fettke J,Lenz C,Albers K,Mallwitz F,Gajovic-Eichelmann N,Ehrentreich-Förster E,Kusch E,Sellrie F

    更新日期:2012-03-31 00:00:00

  • Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding.

    abstract::The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well adapted to each of these substrates was evaluated. Sludge fed with acetate (A), produced a homopolymer of hydroxybutyrate (HB), whereas sludge fed with propionate (P) produced a copolymer of HB and HV (hydoxyvalerate). Switc...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Lemos PC,Serafim LS,Reis MA

    更新日期:2006-03-23 00:00:00

  • Genetical identification of Akebia and Aristolochia species by using Pyrosequencing.

    abstract::This study was performed to determine if Akebia and Aristolochia species (Akebia quinata Decaisne and Aristolochia manshuriensis Kom.) could be identified by genetic analysis of Pyrosequencing method, which is used to assess genetic variation. The Pyrosequencing results of Akebia and Aristolochia species showed differ...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Han S,Kim S,Yang CH,Seo J

    更新日期:2005-12-06 00:00:00

  • Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.

    abstract::Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and ...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Arazi T,Slutsky SG,Shiboleth YM,Wang Y,Rubinstein M,Barak S,Yang J,Gal-On A

    更新日期:2001-04-27 00:00:00

  • High level expression of the bioactive human interleukin-10 in milk of transgenic mice.

    abstract::Human interleukin-10 (hIL-10) has wide spectrum of anti-inflammatory activities and has shown a potential to be used for treatment of inflammatory or immune illness. In this study, transgenic mice that over-express human interleukin-10 (IL-10) in their milk were generated using a bovine beta-casein/human IL-10 hybrid ...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Sohn BH,Chang HG,Kang HS,Yoon H,Bae YS,Lee KK,Kim SJ

    更新日期:2003-06-12 00:00:00

  • Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet's "phosphate crisis".

    abstract::Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe ...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Cho J,Choi K,Darden T,Reynolds PR,Petitte JN,Shears SB

    更新日期:2006-11-01 00:00:00

  • Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production.

    abstract::The Gram-positive Corynebacterium glutamicum is auxotrophic for biotin. Besides the biotin uptake system BioYMN and the transcriptional regulator BioQ, this bacterium possesses functional enzymes for the last three reactions of biotin synthesis starting from pimeloyl-CoA. Heterologous expression of bioF from the Gram-...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Peters-Wendisch P,Götker S,Heider SA,Komati Reddy G,Nguyen AQ,Stansen KC,Wendisch VF

    更新日期:2014-12-20 00:00:00

  • Analysis of reduction of Geobacillus stearothermophilus spores treated with high hydrostatic pressure and mild heat in milk buffer.

    abstract::Our unpublished experimental results of fractional factorial experiments showed that the significant external factors affecting high pressure processing (HPP) inactivation were pressure, temperature and pressure holding time. Based on these results, response surface methodology (RSM) was employed in the present work a...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Gao YL,Ju XR,Jiang HH

    更新日期:2006-09-18 00:00:00

  • Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75.

    abstract::The invariant surface glycoprotein ISG75 is a transmembrane glycoprotein occurring on the surface of the bloodstream-form Trypanozoon. This study describes the expression and purification of the N-terminal extracellular domain of ISG75, a novel target for development of diagnostic tests for trypanosomosis. To facilita...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Tran T,Büscher P,Vandenbussche G,Wyns L,Messens J,De Greve H

    更新日期:2008-06-30 00:00:00

  • I-SceI enzyme mediated integration (SEMI) for fast and efficient gene targeting in Trichoderma reesei.

    abstract::We previously showed that creation of a double strand DNA break (DSB) by expressing I-SceI in an engineered Trichoderma reesei (Hypocrea jecorina) strain containing a I-SceI recognition site improved transformation and homologous integration efficiencies. In this study, we further improved homologous integration frequ...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Ouedraogo JP,Arentshorst M,Nikolaev I,Barends S,Ram AF

    更新日期:2016-03-20 00:00:00

  • Rewiring global regulator cAMP receptor protein (CRP) to improve E. coli tolerance towards low pH.

    abstract::Bioprocesses such as production of organic acids or acid hydrolysis of bioresources during biofuel production often suffer limitations due to microbial sensitivity under acidic conditions. Approaches for improving the acid tolerance of these microbes have mainly focused on using metabolic engineering tools. Here, we t...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Basak S,Geng H,Jiang R

    更新日期:2014-03-10 00:00:00

  • Continuous cultivation start-up control--an experimental investigation.

    abstract::A control strategy to avoid development of synchronous growth in carbohydrate limited Saccharomyces cerevisiae cultivations is proposed and experimentally investigated. The basic idea is to control the metabolic flux through the pathways by manipulating the substrate feed rate to keep the ethanol concentration at a lo...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Møller H,Jørgensen SB

    更新日期:1997-02-28 00:00:00

  • Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I.

    abstract::Hepatocyte growth factor (HGF) is biosynthesized as a biologically inactive, single-chain form (pro-HGF). Its activation is associated with cleavage at Arg494-Val495 into a two-chain mature form composed of disulfide-linked alpha- and beta-chains. Because serum is a major source of HGF activator (the predominant serin...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Hayata D,Fukuta K,Matsumoto K,Adachi E,Hanada K,Adachi K,Nakamura T

    更新日期:2008-02-29 00:00:00

  • Monitoring Chinese hamster ovary cell culture by the analysis of glucose and lactate metabolism.

    abstract::Monitoring cell growth is crucial to the success of an animal cell culture process that can be accomplished by a variety of direct or indirect methodologies. Glucose is a major carbon and energy source for cultured mammalian cells in most cases, but glycolytic metabolism often results in the accumulation of lactate. G...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Tsao YS,Cardoso AG,Condon RG,Voloch M,Lio P,Lagos JC,Kearns BG,Liu Z

    更新日期:2005-08-22 00:00:00

  • Albumin-binding domain from Streptococcus zooepidemicus protein Zag as a novel strategy to improve the half-life of therapeutic proteins.

    abstract::Recombinant antibody fragments belong to the promising class of biopharmaceuticals with high potential for future therapeutic applications. However, due to their small size they are rapidly cleared from circulation. Binding to serum proteins can be an effective approach to improve pharmacokinetic properties of short h...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Cantante C,Lourenço S,Morais M,Leandro J,Gano L,Silva N,Leandro P,Serrano M,Henriques AO,Andre A,Cunha-Santos C,Fontes C,Correia JDG,Aires-da-Silva F,Goncalves J

    更新日期:2017-07-10 00:00:00

  • Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment.

    abstract::In this paper, single-stranded (ss)DNA aptamers with capability to distinguish differentiated PC12 cells from normal PC12 cells were selected by subtractive systematic evolution of ligands by exponential enrichment (SELEX) method. Before each round of selection, randomized ssDNAs were incubated with regular PC12 cells...

    journal_title:Journal of biotechnology

    pub_type: 杂志文章


    authors: Wang C,Zhang M,Yang G,Zhang D,Ding H,Wang H,Fan M,Shen B,Shao N

    更新日期:2003-04-10 00:00:00