Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Library preparation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,189 records · Page 66Linked to original sources
PubMed1998

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases.

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide containing the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized.

Cystine↗
PubMed2004

Preparation of cyclic peptide libraries using intramolecular oxime formation.

A new method for the synthesis of cyclic head-to-side chain peptide libraries has been developed in which the key cyclization step involves reaction between a C-terminal ketone and an N-terminal hydroxylamine to form a macrocyclic oxime. This methodology efficiently delivers cyclic products that consist of mixtures of syn and anti isomers.

Combinatorial Chemistry Techniques↗
PubMed1996

Discovery of a herbicidal lead using polymer-bound activated esters in generating a combinatorial library of amides and esters.

A combinatorial library containing mixtures of amides and esters was prepared through solid-phase chemistry. The advantages of using solid-phase chemistry over solution-phase chemistry to prepare this library are discussed. The library was screened through a high-throughput whole organism herbicidal assay upon which a mixture containing amides was found to have herbicidal activity. Deconvolution of the mixture provided N-(3-benzoylphenyl)-3-(1,1-dimethylethyl)-1-methyl)-1 H-pyrazole-5-carboxamide as a herbicidal lead with broadleaf and narrowleaf pre-emergence herbicidal activity as low as 100 g/ha on some weed species. This study represents the first report of an agrochemical discovered using a combinatorial approach.

Amides↗
PubMed1995

A method for preparing M13 or pUC libraries for sequencing DNA with high G + C or A + T contents.

A simple method is described to generate M13 or pUC libraries from DNA with a very high G + C or A + T content. The G + C-rich DNA is partially digested with HinPI or HpaII restriction enzymes and cloned into the vector linearized in its multiple cloning site with AccI. The A + T-rich DNA is partially digested with TspI and cloned into the EcoRI-linearized vector. These libraries are suitable for large-scale DNA sequencing.

Base Composition↗
PubMed2004

Application of the four-component Ugi condensation for the preparation of sulfated glycoconjugate libraries.

A focused library of novel, sulfated glycoconjugates was synthesized by utilizing carbohydrate-derived blocks in the four-component Ugi condensation. Library members comprise a sulfated monosaccharide linked by various spacers to either an aromatic or monosulfated moiety, or a second sulfated monosaccharide. The affinities of these heparan sulfate (HS) mimetics for the HS-binding fibroblast growth factors FGF-1 and FGF-2 were measured via a surface plasmon resonance solution affinity assay.

Fibroblast Growth Factor 1↗
PubMedDate not supplied

Solid-phase directed ortho-lithiation and the preparation of a phthalide library.

An efficient solid-phase synthesis of phthalides is described in which aromatic carboxylic acids or acid chlorides and ketones are used as building blocks. The carboxylic acid or acid chloride is tethered to aminomethylated polystyrene resin, forming a secondary amide, which functions as both the linker and the directing metalation group. This allows the resin-bound benzamides to be ortho-lithiated at 0 degrees C. The ortho-lithiated species can be quenched with benzaldehydes, benzophenones, and even acetophenones, affording resin-bound alcohols. A cyclative cleavage is induced by simply warming the resin in toluene or dioxane, yielding the desired phthalide compounds in exceptionally high purity.

Journal Article↗
PubMedDate not supplied

Copper (II)-mediated arylation with aryl boronic acids for the N-derivatization of pyrazole libraries.

A N-derivatized 3-dimethylaminopropyloxypyrazole library was prepared using solution-phase parallel synthesis. The library was designed using physicochemical constraints designed to remove non-membrane-permeable molecules. Cupric acetate-mediated N-arylation with aryl boronic acids proceeded regioselectively to form the N-2-substituted derivatives. The presence of the 3-dimethylaminopropyloxy group was found to completely control the regioselectivity of the arylation. Presence of a dimethylaminoethyloxy or dimethylaminobutyloxy group gave a lesser degree of regioselectivity. The scope of the method as applied to library synthesis is discussed.

Journal Article↗
PubMed1997

Limitations of the coupling of amino acid mixtures for the preparation of equimolar peptide libraries.

The standard method of peptide library synthesis involves coupling steps in which a single amino acid is reacted with a mixture of resin-bound amino acids. The more recently described positional scanning strategy (in which each position in the peptide sequence is occupied in turn by a single residue) is different since it involves the coupling of mixtures of amino acids to mixtures of resin-bound amino acids. In the present study, we analyze the compounds produced under these conditions measuring coupling rates and amounts of formed products, using mainly UV, HPLC, LC/MS and MS/MS techniques. Our data do not permit to conclude that the resulting libraries are complete. Indeed, our analytical data indicate that a large part of the di-, tri- and tetrapeptides synthesized with this method are not present in the final mixture. Although chemical compensation (in which poor coupling kinetics is compensated by a larger excess of the incoming amino acid) has been thought to counterbalance these biases, our experiments show that the compensation method does not take into account the crucial influence of the resin-bound amino acid and that even the dipeptide libraries obtained in this way are far from completeness. The present work provides strong evidence that the coupling of mixtures of amino acids to resin-bound residues, which is required by the positional scanning strategy, results in incomplete and/or non-equimolar libraries. It also clearly confirms that coupling rates in solid-phase peptide synthesis are dependent on the nature of both the incoming and the immobilized amino acid.

Amino Acids↗
PubMed1984

Molecular cloning of a cDNA sequence complementary to porphobilinogen deaminase mRNA from rat.

A cDNA clone containing sequences complementary to the mRNA coding for anemic rat spleen porphobilinogen deaminase (EC 4.3.1.8) has been isolated. A cDNA library was prepared from partially purified mRNA (1% purity). This library was then screened by colony hybridization, using a cDNA probe derived from porphobilinogen deaminase mRNA further enriched (10-20% purity) by gel electrophoresis in the presence of methylmercury hydroxide. Colonies hybridizing with the probe were analyzed by hybrid-selected translation using anemic rat spleen mRNA. Four recombinant plasmids containing porphobilinogen deaminase cDNA sequences were identified by specific immunoprecipitation of the translational product from hybrid-selected mRNA. Porphobilinogen deaminase mRNA was shown to contain 1800 bases by blot hybridization analysis. The cloned cDNA sequence consists of 1500 bases. Hybridization analysis of poly(A)+ RNA from uninduced and induced mouse erythroleukemic cells indicated that induction to erythroid differentiation by dimethyl sulfoxide results in a 10-fold increase of porphobilinogen deaminase mRNA. The rat cDNA clones hybridize to the corresponding sequences encoding human porphobilinogen deaminase. This property will be useful for isolation of human gene(s) and further characterization of the molecular lesion(s) responsible for acute intermittent porphyria.

Ammonia-Lyases↗
PubMed1999

Preparation of a genomic library using a TA vector.

An efficient and simple method for constructing a genomic DNA library is presented using a TA cloning vector. It is based on sonication cleavage of genomic DNA, blunting of the fragment ends with mung bean nuclease, and addition of a single 3'-deoxyadenylate with Taq DNA polymerase, followed by ligation with a TA vector. This method is useful for improving the quality of genomic libraries for organisms whose genomic DNA is not well digested with restriction enzymes owing to the presence of polysaccharides and/or DNA methylation.

Alkyl and Aryl Transferases↗
PubMed1983

Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene.

Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.

Animals↗
PubMed1997

Preparation of oligosaccharide units library and its utilization.

There is high current interest in developing synthetic routes to oligosaccharides involved in glycoconjugates. Significant attention has been focused on the application of glycosidase-catalyzed transglycosylation for practical synthesis of oligosaccharides. The enzymatic synthesis has become more practical by the use of several glycosidases available in sufficient quantities. This review describes convenient syntheses of di- and trisaccharide units, which are related to molecular recognition, by using regioselective transgalactosylation, trans-N-acetylglucosaminylation, transfucosylation, and transmannosylation. The regioselectivity could be controlled to some extent by using the following techniques: (1) varying enzymes, (2) organic co-solvent system, (3) the configuration of the existing glycosidic linkage of the acceptor and (4) inclusion complex of acceptor glycoside with cyclodextrin. Furthermore, glycopolymers carrying a series of disaccharides containing beta-D-galactosyl residues were synthesized and used as a model in oligosaccharide-lectin interaction analysis. These water-soluble glycopolymers were shown to be useful as probes of carbohydrate recognition.

Acetylglucosamine↗
PubMed2003

[Isolation of differentially expressed genes in human osteoblast-like osteosarcoma cell line induced with 17beta-estradiol].

OBJECTIVE: To obtain a serial differentially expressed cDNA fragments from human osteoblast-like osteosarcoma MG-63 cells induced with 17beta-estradiol and to find some estrogen-responsive genes. METHODS: Optimized cDNA representational difference analysis (RDA) was performed to isolate up-regulated expressed sequences between cDNA from MG-63 cell line treated with and without 17beta-estradiol. The sources of up-regulated expressed cDNA fragments were proved by Southern blot. The fragments were cloned into the pGEM-T easy vector and a cDNA library was prepared in E. coli JM109 cells. The cDNA library was plated on LB/Amp(+)/X-gal/IPTG plates and white colonies were picked up and individually grown in LB/Amp(+) medium in 96-well plates. After PCR, colonies were individually blotted onto a Hybond N membrane. Membranes were hybridized with alpha-(32)P-labeled subtracted or unsubtracted tester cDNA. Clones showing a strong hybridization signal with the forward-subtracted probes compared with the reverse-subtracted ones were selected for DNA sequencing, BLAST and Northern blot analysis after release of the pGEM-T easy insert with EcoR I. RESULTS: Five up-regulated expressed fragments were isolated in the fourth subtraction hybridization using cDNA from MG-63 cells induced with 17beta-estradiol as tester amplicon and cDNA from untreated MG-63 cells as driver amplicon by cDNA RDA. These fragments were proved to be really coming from tester amplicon and down-regulated expressed in the untreated cell by Southern blotting. We obtained more than 600 cDNA clones with positive insert from MG-63 cells line induced with 17beta-estradiol and about 120 differentially expressed clones through dot blotting. Twenty clones were sequenced and we got 15 gene sequences. One of them was proved to be differentially expressed through Northern blotting. CONCLUSIONS: cDNA RDA is one of the most effective methods which can isolate differentially expressed genes and we can screen differentially expressed genes rapidly through cDNA RDA combined with cDNA arrays. Some genes of human osteoblast-like osteosarcoma MG-63 cell line showed up-regulated expression after induction by 17beta-estradiol.

Blotting, Northern↗