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 1,171 records · Page 65Linked to original sources
PubMed1999

Preparation of a cDNA library and preliminary assessment of 1400 genes from mouse growth cartilage.

Cartilage is an inconvenient tissue for the isolation of mRNA, and this has hampered studies of its component mRNAs conducted to date. Here, we describe the preparation of a good quality cDNA library from mouse growth cartilage (mGC). A total of 1.7 microg of poly(A)+ RNA was obtained from about 1200 pieces of the mGC zone of 60 young mice (BALB/c, 4 weeks old). Using this poly(A)+ RNA, we constructed a cDNA library using the pAP3neo vector by the linker-primer method. The complexity of the cDNA library was 2.6 x 106 colony-forming units (cfu), which signified that almost all of the mRNA components in the mGC were present in this cDNA library. From this library, 1401 clones were randomly selected and their insert sizes were examined. Of these clones, 166 (12%) had no inserts, 466 (33%) had inserts ranging in size from 0-0.9 kbp, 480 (34%) had inserts of 1. 0-1.9 kbp, 162 (12%) had inserts of 2.0-2.9 kbp, and 127 (9%) had sizes greater than 3.0 kbp. The average insert size was 1.45 kbp. The number of cfu and the insert size data qualified this library as of reasonably good quality. Clones with an insert size greater than 1 kbp (769 clones) were sequenced from their 5' ends. Among the 769 clones examined, 608 gave sequence data. Among these, 196 (32%) were unknown, 2 were only poly A, and 410 (67%) coded for known proteins. Of these, 55 clones coded for type II (pro)collagen, 54 for osteonectin, and 22 for other cartilage collagens (type IX, type X, and type XI). The rest included cartilage extracellular matrix genes, general cellular genes, and others. To judge further the quality of the library, 45 species coding for type II collagen chain were aligned based on their 5' end sequences. Three species (7%) contained almost the full-length insert, and the shortest one was 1. 5 kbp in length (full-length 5.6 kbp). These data show that this cDNA library is of reasonably good quality, making it likely that the large number of unknown inserts (32%) will provide a suitable pool for the identification and functional determination of new GC genes.

Animals↗
PubMed1998

Kaleidaseq: a Web-based tool to monitor data flow in a high throughput sequencing facility.

Tracking data flow in high throughput sequencing is important in maintaining a consistent number of successfully sequenced samples, making decisions on scheduling the flow of sequencing steps, resolving problems at various steps and tracking the status of different projects. This is especially critical when the laboratory is handling a multitude of projects. We have built a Web-based data flow tracking package, called Kaleidaseq, which allows us to monitor the flow and quality of sequencing samples through the steps of preparation of library plates, plaque-picking, preparation of templates, conducting sequencing reactions, loading of samples on gels, base-calling the traces, and calculating the quality of the sequenced samples. Kaleidaseq's suite of displays allows for outstanding monitoring of the production sequencing process. The online display of current information that Kaleidaseq provides on both project status and process queues sorted by project enables accurate real-time assessment of the necessary samples that must be processed to complete the project. This information allows the process manager to allocate future resources optimally and schedule tasks according to scientific priorities. Quality of the sequenced samples can be tracked on a daily basis, which allows the sequencing laboratory to maintain a steady performance level and quickly resolve dips in quality. Kaleidaseq has a simple easy-to-use interface that allows access to all major functions and process queues from one Web page. This software package is modular and designed to allow additional processing steps and new monitoring variables to be added and tracked with ease. Access to the underlying relational database is through the Perl DBI interface, which allows for the use of different relational databases. Kaleidaseq is available for free use by the academic community from http://www.cshl.org/kaleidaseq.

Arabidopsis↗
PubMed1998

An improved approach for construction of bacterial artificial chromosome libraries.

Presented here are improved methodologies that enable the generation of highly redundant bacterial artificial chromosome/P1-derived artificial chromosome libraries, with larger and relatively uniform insert sizes. Improvements in vector preparation and enhanced ligation conditions reduce the number of background nonrecombinant clones. Preelectrophoresis of immobilized high-molecular-weight DNA removes inhibitors of the cloning process, while sizing DNA fragments twice within a single gel effectively eliminates small restriction fragments, thus increasing the average insert size of the clones. The size-fractionated DNA fragments are recovered by electroelution rather than the more common melting of gel slices with subsequent beta-agarase treatment. Concentration of the ligation products yields a 6- to 12-fold reduction in the number of electroporations required in preparing a library of desirable size. These improved methods have been applied to prepare PAC and BAC libraries from the human, murine, rat, canine, and baboon genomes with average insert sizes ranging between 160 and 235 kb.

Animals↗
PubMed1992

Preparation of soluble peptide libraries: application to studies of platelet adhesion sequences.

We describe chemical synthesis of peptide mixtures that equally express many sequence combinations. Using 65 couplings of single amino acids, five mixtures were prepared with the sequences Tyr-Gly-Arg-Gly-Yyy-Xxx-Xxx, where Yyy is Ser, Asp, Arg, Asn, or Glu, and Xxx is any amino acids. Compositional and sequence analyses supported full representation of all amino acids, except isoleucine was deficient in the sixth position. The data suggest formation of a repertoire of 1,900 sequence combinations (5 x 19 x 20). The mixture with Asp as the fifth residue inhibited platelet adhesion to fibronectin more effectively than the other mixtures. Peptide libraries offer a new tool for investigating bioactive peptides.

Amino Acid Sequence↗
PubMed2005

Sorghum expressed sequence tags identify signature genes for drought, pathogenesis, and skotomorphogenesis from a milestone set of 16,801 unique transcripts.

Improved knowledge of the sorghum transcriptome will enhance basic understanding of how plants respond to stresses and serve as a source of genes of value to agriculture. Toward this goal, Sorghum bicolor L. Moench cDNA libraries were prepared from light- and dark-grown seedlings, drought-stressed plants, Colletotrichum-infected seedlings and plants, ovaries, embryos, and immature panicles. Other libraries were prepared with meristems from Sorghum propinquum (Kunth) Hitchc. that had been photoperiodically induced to flower, and with rhizomes from S. propinquum and johnsongrass (Sorghum halepense L. Pers.). A total of 117,682 expressed sequence tags (ESTs) were obtained representing both 3' and 5' sequences from about half that number of cDNA clones. A total of 16,801 unique transcripts, representing tentative UniScripts (TUs), were identified from 55,783 3' ESTs. Of these TUs, 9,032 are represented by two or more ESTs. Collectively, these libraries were predicted to contain a total of approximately 31,000 TUs. Individual libraries, however, were predicted to contain no more than about 6,000 to 9,000, with the exception of light-grown seedlings, which yielded an estimate of close to 13,000. In addition, each library exhibits about the same level of complexity with respect to both the number of TUs preferentially expressed in that library and the frequency with which two or more ESTs is found in only that library. These results indicate that the sorghum genome is expressed in highly selective fashion in the individual organs and in response to the environmental conditions surveyed here. Close to 2,000 differentially expressed TUs were identified among the cDNA libraries examined, of which 775 were differentially expressed at a confidence level of 98%. From these 775 TUs, signature genes were identified defining drought, Colletotrichum infection, skotomorphogenesis (etiolation), ovary, immature panicle, and embryo.

DNA, Complementary↗
PubMed1998

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library.

BACKGROUND: Osteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast-specific biological functions remain elusive because only a limited number of osteoclast-specific genes have been identified. To circumvent this, we isolated a large number of osteoclast-specific genes by preparing a subtracted cDNA library of high quality. RESULTS: We first constructed a plasmid expression vector (pAP3neo) that allowed an efficient subtraction. Then, we improved the standard protocols for preparation of the cDNA library and the subsequent subtraction procedure. Using our protocol, we prepared a rabbit osteoclast cDNA library of high complexity. Subsequently, we prepared an osteoclast-specific cDNA library of high complexity by subtracting it with biotin-labelled mRNA, derived from rabbit spleen through the biotin-avidin method. The resulting library included a high proportion of full-length cDNA inserts. Using DNA dot blot analysis, we found that the osteoclast-specific cDNA clones were highly enriched in this subtracted cDNA library, i.e. nearly 70% of the analysed clones were primarily detected in osteoclasts but not in spleen. Multiple-tissue Northern analysis also showed that many of these clones were expressed almost exclusively in osteoclasts. DNA sequencing of randomly selected clones showed that 424 cDNA species out of 1136 analysed were novel. DNA sequencing also showed that our subtracted cDNA library was almost equalized, suggesting that the library may contain almost all of the osteoclast-specific genes. CONCLUSION: From these data, we conclude that our subtraction protocols, and the subsequent procedure for the analysis of the isolated clones developed here, are useful for the comprehensive isolation and identification of transcriptionally up- or down-regulated genes in general.

Animals↗
PubMed1996

The synthesis of peptidomimetic combinatorial libraries through successive amide alkylations.

A soluble peptidomimetic combinatorial library of 57,500 compounds was prepared. This library has a dipeptide scaffold with each amide hydrogen replaced with five different alkyl groups (methyl, ethyl, allyl, benzyl, or naphthylmethyl). Solid-phase methodology in combination with N-alkylation were used to synthesize the library, which incorporated 50 different L-, D-, and unnatural amino acids. Repetitive amide alkylations were carried out on the solid support following each amino acid coupling step. Individual model compounds were synthesized in order to optimize the alkylation conditions, to study potential amino acid side chain modifications, to determine the extent of racemization, and to provide analytical controls during the library synthesis.

Alkylation↗
PubMed2004

Humanization of agonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library.

Given the key role 4-1BB plays in the stimulation of T cells, humanization of agonistic anti-human 4-1BB monoclonal antibody (mAb) may have important clinical applications. In this paper, we present the humanization of agonistic anti-human 4-1BB mAb, BBK-4, using a phage display library. We first prepared the combinatorial library by incorporating murine and human alternative at positions representing buried residues that might affect the structural integrity of the antigen binding site. Six humanized single chain Fv (scFv) fragments were selected from the combinatorial library expressing phage-displayed humanized scFv. They were found to retain the epitope specificity of the original mAb but had affinities of lower than 1/10 of the original. In spite of the lower affinity, the humanized scFv coated on the surface expanded human peripheral blood mononuclear cells (PBMCs) in MLR similarly to the original mAb in the presence of anti-CD3 mAb. These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application.

Amino Acid Sequence↗
PubMed2001

Isolation of 105 microsatellite loci from an ovine genomic library enriched for microsatellites.

This paper reports on the development of a small-insert (approximately 700 bp) total-genomic library for sheep specifically designed for enrichment for microsatellite (ms) loci. Four enriched libraries were prepared by amplification of the primary library with CA15, CA11, TG15 and TG11 oligonucleotide primers. A total of 11,020 clones was recovered, screened for dinucleotide repeats and over 500 positive clones sequenced. Sequence analysis indicated low clone redundancy and yielded 105 new ovine ms loci. Seventy-two percent of the new loci were found to be polymorphic in the sires of the AgResearch International Mapping Flock (IMF). The 105 new microsatellite loci increase the number of microsatellites available for sheep by >7%.

Animals↗
PubMed1998

Laminar shear stress-induced GRO mRNA and protein expression in endothelial cells.

BACKGROUND: The shear stress induced by blood flow may play a pivotal role in the induction or prevention of atherosclerosis by changing endothelial functions. To disclose the mechanisms of this change, we prepared an endothelial cell (EC) cDNA library to select specific clones expressed in response to shear stress. METHODS AND RESULTS: The mRNA of cultured confluent bovine aortic ECs (BAECs) subjected to steady laminar shear stress (30 dyne/cm2) for 4 hours was separated, and a cDNA library was prepared. Nine clones whose expressions were specifically enhanced by the shear stress were selected by use of a differential hybridization method. One clone had 94% homology at the nucleotide sequence level to Oryctolagus cuniculus gro (GRO) mRNA and 79% homology at the amino acid sequence level to human GRO-beta. The GRO mRNA expression was increased in both BAECs and human umbilical vein ECs (HUVECs) after the ECs were subjected to high (30 dyne/cm2) and low (5 dyne/cm2) laminar shear stress. GRO-alpha and/or -beta protein expression also increased after the HUVECs and BAECs were subjected to shear stress. Because GRO protein has been shown to function as an adhesion factor of monocytes on the surface of ECs, we studied whether shear stress-induced monocyte adhesion was caused by GRO protein expression on ECs. The 4-hour shear stress enhanced monocyte adhesion to ECs by 2.5-fold over control levels, and this enhancement was inhibited by 53% by anti-GRO-alpha antibody. CONCLUSIONS: The present study is the first report that shear stress induced the expression of GRO mRNA and protein in ECs and enhanced the monocyte adhesion on ECs via GRO protein. Further investigations of the functions and participation in atherogenesis of this selected clone may clarify the significance of shear stress on atherogenesis.

Animals↗
PubMed1987

DNA probes for detection and identification of mycoplasmas (Mollicutes).

DNA probes are expected to prove a specific, sensitive, rapid and inexpensive means for diagnosis of mycoplasma infections, replacing procedures that depend on cultivation of the fastidious organisms. Probes made up of conserved genes, such as rRNA genes, do offer the advantage of identifying and distinguishing multiple species with a single labeled reagent. The mycoplasmal rRNA gene probe pMC5 was effective in detection and identification of mycoplasmas infecting cell cultures. However, use of pMC5 for detection of spiroplasmas and mycoplasma-like organisms (MLOs) in infected plants was hindered by hybridization of this probe with chloroplast rRNA genes. Moreover, for identifying species and strains by pMC5, a complex hybridization procedure--involving DNA purification, digestion, electrophoresis, and Southern blot hybridization--is required. More specific DNA probes, on the other hand, can identify specific Mollicutes by the much simpler, faster and more sensitive dot blot technique. Thus, a probe made of a cloned Spiroplasma citri plasmid could detect by this technique as little as 10 pg of S. citri DNA (equivalent to about 10(3) organisms) in infected plants and insects. DNA probes specific for Mycoplasma pneumoniae and M. genitalium were selected from genomic libraries and prepared in pUC13 by screening the libraries for inserts hybridizing only with DNA of the specific mycoplasma. The probes, labeled by nick translation with 32P-nucleotides, could detect as little as approximately 100 pg of the specific mycoplasmal DNA by dot blot hybridization. To eliminate radioactivity, the above DNA probes were labeled by biotinylation of sulfonation systems. Dot blot hybridization with these probes showed decreased sensitivity of detection by about one order of magnitude, and some nonspecific background reaction with large quantities of nonhomologous DNAs.

Animals↗
PubMed1993

Peptide libraries: criteria and trends.

The development of approaches for preparing peptide libraries, containing millions of different amino acid sequences of a specified length, provides an invaluable resource for characterizing the molecular interactions that underlie many biological processes. Such libraries can also be used for identifying novel sequences that have a desired biological activity.

Amino Acid Sequence↗
PubMed2025

Whole genome sequencing analysis of Candida glabrata isolates collected from patients with selected drug-resistant candidiasis hospitalized in Eastern Poland.

The epidemiology data for candidiasis indicate an increase in Candida glabrata infections. Moreover, several reports have shown an increasing number of drug-resistant cases of these infections. The source of drug resistance can often be traced to genetic mutations in genes related to a drug's mechanism of action. Therefore, we conducted whole genome sequencing of several drug-resistant isolates of Candida glabrata collected from patients hospitalized in Eastern Poland to assess whether mutations in selected genes correlated with susceptibility analysis results. The fungal species from patient samples were identified, and the isolated Candida glabrata were subjected to antifungal drug susceptibility testing. The results were interpreted according to the EUCAST and CLSI recommendations. Susceptibility to 5-flucytosine was assessed using the ATB FUNGUS kit. Libraries were prepared according to the NEXTERA XT DNA Library Prep and subsequently sequenced. The outcomes indicated common resistance to two of the three analyzed echinocandins, as well as two cases of simultaneous resistance to echinocandins and selected azole-based drugs. We detected several previously reported mutations in selected resistance-related genes, as well as five that are first described here: ERG5 (M267I), ERG6 (R57K), PDH1 (K438Q, V434I, F600V, V1192S), FCY1 (M129T), and FCY2 (I384F). Neither of the identified nonsynonymous mutations was correlated with the drug resistance demonstrated in the susceptibility testing. Furthermore, we can exclude the possibility of acquired drug resistance, thereby raising questions about the possibility of unknown mechanisms of resistance to azole-based and echinocandin drugs.

Candida glabrata↗
PubMed1995

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11.

Bovine pancreatic ribonuclease A (RNase A) has been the object of much landmark work in biological chemistry. Yet the application of the techniques of protein engineering to RNase A has been limited by problems inherent in the isolation and heterologous expression of its gene. A cDNA library was prepared from cow pancreas, and from this library the cDNA that codes for RNase A was isolated. This cDNA was inserted into expression plasmids that then directed the production of RNase A in Saccharomyces cerevisiae (fused to a modified alpha-factor leader sequence) or Escherichia coli (fused to the pelB signal sequence). RNase A secreted into the medium by S.cerevisiae was an active but highly glycosylated enzyme that was recoverable at 1 mg/l of culture. RNase A produced by E.coli was in an insoluble fraction of the cell lysate. Oxidation of the reduced and denatured protein produced active enzyme which was isolated at 50 mg/l of culture. The bacterial expression system is ideal for the large-scale production of mutants of RNase A. This system was used to substitute alanine, asparagine or histidine for Gln11, a conserved residue that donates a hydrogen bond to the reactive phosphoryl group of bound substrate. Analysis of the binding and turnover of natural and synthetic substrates by the wild-type and mutant enzymes shows that the primary role of Gln11 is to prevent the non-productive binding of substrate.

Amino Acid Sequence↗
PubMed1988

DNA probes for mycobacteria. I. Isolation of DNA probes for the identification of Mycobacterium tuberculosis complex and for mycobacteria other than tuberculosis (MOTT).

Traditional methods used in identifying mycobacteria such as acid-fast bacillus stains and culture are often time-consuming, insensitive and non-specific. The isolation of DNA probes, coupled to a non-radioactive, e.g. biotin-based detection system, have the potential to foster the development of clinical assays for Mycobacterium tuberculosis and mycobacteria other than tuberculosis (MOTT) that are rapid, sensitive and specific. To this end, we have isolated two different probes: one which is specific for the Mtb complex and one which recognizes all other potentially pathogenic mycobacteria. The use of these probes in combination should allow the detection and differentiation of M. tuberculosis from MOTT. To isolate the first probe, we prepared a library of M. tuberculosis DNA fragments in a lambda EMBL phage vector. Recombinant phage were screened by plaque-lift hybridization procedures using nick-translated mycobacterial genomic DNA to identify sequences specific to the Mtb complex. Inserts from candidate recombinant phage were purified, nick-translated and hybridized against a wide variety of filter-bound mycobacterial and non-mycobacterial DNAs. Two clones were identified which hybridized to the closely related M. tuberculosis, M. bovis and M. microti but not to other species of mycobacteria. The second probe was isolated by preparing a library of M. malmoense DNA fragments in lambda EMBL and screening by plaque-lift hybridization. One clone was identified which, in addition to recognizing members of the Mtb complex, also hybridized to M. intracellulare, M. malmoense, M. scrofulaceum, M. simiae, M. xenopi, M. avium, M. szulgai, M. kansasii and M. haemophilum. None of the three clones hybridized to DNA from non-mycobacterial species.

Animals↗
PubMed1992

Use of large-scale hydrazinolysis in the preparation of N-linked oligosaccharide libraries: application to brain tissue.

In this report, we describe the preparation of a library of N-linked glycans from whole murine brain obtained by the large-scale hydrazinolysis of an acetone powder of the tissue followed by chromatographic procedures. 84% of the characterized oligosaccharides were found to be anionic, the remainder neutral. The anionic species were successively neutralized by neuraminidase (29%), aq. hydrofluoric acid (30%), and methanolysis (26%), indicating that approximately equal portions were sensitive to desialylation, dephosphorylation and desulfation, respectively. The presence of the sulfated fraction was confirmed by direct 35SO4 metabolic labelling. A residual partially characterized fraction was found to be anionic through possession of carboxylic acid groups, unrelated to sialic acid. The purified oligosaccharides, in the absence of their original protein conjugates, were shown to retain those immunological characteristics essential for recognition by a specific monoclonal antibody, LS (412), that is known to recognize a carbohydrate epitope present on a number of neural adhesion molecules and functional in neural cell adhesion. These properties confirm the viability of scaling up the size of the hydrazinolysis procedure and adapting it to whole tissue for the production of glycan libraries and for the probing of structures of interest.

Animals↗
PubMed1988

Isolation of Salmonella typhimurium cys genes by transduction with a library of recombinant plasmids packaged in bacteriophage P22HT capsids.

We have prepared a library of Salmonella typhimurium genomic fragments cloned in pBR322 and packaged in P22HT capsids. Plasmids carrying 24 of 26 specific genes searched for were isolated by transduction at frequencies of 1 to 344 per 10(6) plasmid transductants. All 11 known genes of the cysteine regulon were isolated from this library, including cysK, which we had previously been unable to clone in a recombinant plasmid with an Escherichia coli host. This library provides a simple and rapid method for isolating most S. typhimurium genes by using S. typhimurium itself as a host and should be particularly useful for cloning genes that might be deleterious to E. coli.

Bacteriophages↗
PubMed1994

Adaptor-based uracil DNA glycosylase cloning simplifies shotgun library construction for large-scale sequencing.

An improved strategy for the preparation of libraries for the random sequencing of DNA is reported. The protocol is a modification of a previous adaptor-based strategy, and utilizes long (11 base) overhangs, which eliminates the unreliable step of vector-insert ligation. The random inserts are prepared by adaptor ligation, while the M13 vector is prepared as described for uracil DNA glycosylase (UDG) cloning of polymerase chain reaction (PCR) products, using PCR with uracil-containing primers, followed by UDG treatment to produce overhangs. This method has been found to reliably yield large numbers of clones. There is no background due to religation of the vector, and all clones contain inserts. In addition, the method is simple and suitable for export to other investigators. Libraries were constructed from cosmids containing human DNA and from human cDNAs in order to characterize a strategy for shotgun sequencing of multiple shorter fragments.

Base Sequence↗