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 1,333 records · Page 74Linked to original sources
PubMed1985

Isolation and regional mapping of random X sequences from distal human X chromosome.

Chromosome-mediated gene transfer (CMGT) lines were shown to be convenient donors of genomic sequences from specific regions of the genome adjacent to selectable markers. Two libraries were prepared from CMGT lines carrying sequences spanning the long arm of the human X chromosome from HPRT (Xq26) to G6PD (Xq28). A series of 22 CMGT lines sharing the same selectable marker (HPRT) were used in conjunction with five standard translocation hybrids to provide fine-resolution regional mapping of the nonrepetitive X specific probes isolated from the libraries. The order of three human recombinant sequences with respect to known X-linked markers is: PGK (Xq13), 05-02 (DXS78); HPRT (Xq26), 07-03 (DXS79); surface antigen S11 (Xq27), 07-14 (DXS80); and G6PD (Xq28).

Animals↗
PubMed2004

Antimicrobial peptides discovered in the black tiger shrimp Penaeus monodon using the EST approach.

Two cDNA libraries were prepared from hemocytes of normal and Vibrio harveyi-challenged black tiger shrimp Penaeus monodon. A total of 1062 expressed sequence tag (EST) clones were sequenced unidirectionally. ESTs representing the antimicrobial peptide (AMP) homologues, antilipopolysaccharide factors (ALF), penaeidins and crustins were discovered. They predominated among immune-related genes, representing 29.2% and 64.0% of the normal and challenged libraries, respectively. Several types of each AMP homologue were found. Sequence alignments of ALF in P. monodon (ALFPm 1 to 5) implied possible alternative splicing of different exons at both NH2 and COOH-termini. Only one major type of penaeidin (penPm3) was found in P. monodon. In addition, crustin homologues (crusPms1 to 4) and a newly identified glycine-rich antibacterial peptide (GAMPPm1) were also isolated and characterized. Using RT-PCR analysis, expression of ALF, penaeidin and crustin transcripts was detected in various tissues but the main expression site was in hemocytes. Expression of these antimicrobial peptides in P. monodon subjected to V. harveyi challenge revealed a significant increase in expression of ALFPms (p < 0.05) but a decrease in expression of crustins and penaeidins.

Alternative Splicing↗
PubMed1993

Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries.

A large number (33) of human Fab fragments reacting with HIV-1 surface glycoprotein gp120 have been generated by selection from a combinatorial IgG1 kappa library displayed on the surface of phage. The library was prepared from a long term asymptomatic HIV-seropositive donor. Analysis of the sequences from these Fabs shows the heavy chains can be placed in groups, many of which contain intraclonal variants, almost certainly corresponding to chains used in vivo. Further variants can be accessed via chain shuffling experiments in which a given light chain is recombined with a library of heavy chains. Heavy chain promiscuity, i.e. the ability of heavy chains to pair with different light chains with retention of antigen binding, is dependent on the particular heavy chain considered and probably excludes the identification of in vivo light chain partners. The antibodies examined here are primarily to the CD4 binding site on gp120 and broadly reflect the serum profile of the donor. The antibodies show evidence of extensive somatic modification indicative of an antigen-driven response. The heavy chain CDR3 regions of the antibodies show a remarkably conserved extended length. A number also show strong sequence conservation in CDR3 against a background of considerable diversity in the rest of the VH gene supporting a central role for this region in antigen recognition.

Amino Acid Sequence↗
PubMed2004

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides for defining substrate specificity of the angiotensin I-converting enzyme and development of selective C-domain substrates.

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides were used for the analyses of the S(3) to S(1)' subsites of the somatic angiotensin I-converting enzyme (ACE). Substrate specificity of ACE catalytic domains (C- and N-domains) was assessed in an effort to design selective substrates for the C-domain. Initially, we defined the S(1) specificity by preparing a library with the general structure Abz-GXXZXK(Dnp)-OH [Abz = o-aminobenzoic acid, K(Dnp) = N(epsilon)-2,4-dinitrophenyllysine, and X is a random residue], where Z was successively occupied with one of the 19 natural amino acids with the exception of Cys. The peptides containing Arg and Leu in the P(1) position had higher C-domain selectivity. In the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH, Arg was fixed at P(1) so we could define the C-domain selectivity of the S(1)', S(2), and S(3) subsites. On the basis of the results from these libraries, we synthesized peptides Abz-GVIRFK(Dnp)-OH and Abz-GVILFK(Dnp)-OH which contain the most favorable residues for C-domain selectivity. Systematic reduction of the length of these two peptides resulted in Abz-LFK(Dnp)-OH, which demonstrated the highest selectivity for the recombinant ACE C-domain (k(cat)/K(m) = 36.7 microM(-1) s(-1)) versus the N-domain (k(cat)/K(m) = 0.51 microM(-1) s(-1)). The substrate binding of Abz-LFK(Dnp)-OH with testis ACE using a combination of conformational analysis and molecular docking was examined, and the results shed new light on the binding characteristics of the enzyme.

Amino Acid Sequence↗
PubMed2004

Synthesis of nucleoside libraries on solid support. II. 2,6,8-Trisubstituted purine nucleosides using 8-bromoguanosine as precursor.

A series of 2,6,8-trisubstituted purine nucleoside libraries was prepared by parallel solid-phase synthesis using 8-bromoguanosine as a common synthetic precursor. Polystyrene-methoxytrityl chloride resin was linked to the N2 or O5' position of the guanosine analogues. 8-Bromoguanosine was derivatized at the C8 position via carbon-carbon bond formation. Nucleophilic aromatic substitution at C2 and/or C6 positions with various amines produced two series of purine nucleoside libraries with very diverse substitution.

Guanosine↗
PubMed2003

Chlamydomonas reinhardtii genome project. A guide to the generation and use of the cDNA information.

The National Science Foundation-funded Chlamydomonas reinhardtii genome project involves (a) construction and sequencing of cDNAs isolated from cells exposed to various environmental conditions, (b) construction of a high-density cDNA microarray, (c) generation of genomic contigs that are nucleated around specific physical and genetic markers, (d) generation of a complete chloroplast genome sequence and analyses of chloroplast gene expression, and (e) the creation of a Web-based resource that allows for easy access of the information in a format that can be readily queried. Phases of the project performed by the groups at the Carnegie Institution and Duke University involve the generation of normalized cDNA libraries, sequencing of cDNAs, analysis and assembly of these sequences to generate contigs and a set of predicted unique genes, and the use of this information to construct a high-density DNA microarray. In this paper, we discuss techniques involved in obtaining cDNA end-sequence information and the ways in which this information is assembled and analyzed. Descriptions of protocols for preparing cDNA libraries, assembling cDNA sequences and annotating the sequence information are provided (the reader is directed to Web sites for more detailed descriptions of these methods). We also discuss preliminary results in which the different cDNA libraries are used to identify genes that are potentially differentially expressed.

Animals↗
PubMed2003

Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles.

Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.

Avidin↗
PubMed1998

Excellence, promise, vision, and values: reflections on the Janet Doe Lectures, 1967-1997.

As the Medical Library Association prepares to enter its second century of service to society, it can look back with pride on the remarkable record of achievements of the association and its members, both personal and institutional, which are preserved for posterity in the Janet Doe Lectures. Established in 1966 by an anonymous donor, the Janet Doe Lectures on the history or philosophy of medical librarianship trace the development of health sciences libraries and librarianship and the evolution of the Medical Library Association. The major themes which emerge from this comprehensive review of the lectures published between 1967 and 1997 include the changing roles and values of health sciences libraries and librarianship; education and credentialing of health sciences librarians; MLA activities and concerns; and MLA/National Library of Medicine relations.

Certification↗
PubMed1999

Generation of native bovine mAbs by phage display.

Modeling of disease pathogenesis and immunity often is carried out in large animals that are natural targets for pathogens of human or economic relevance. Although murine mAbs are a valuable tool in identifying certain host/pathogen interactions, progress in comparative immunology would be enhanced by the use of mAbs isolated from the host species. Such antibodies would reflect an authentic host immune response to infection or vaccination, and as they are host derived, would allow the application of in vivo experiments that previously have been unrealizable in large animals because of induction of an antispecies immune response. The advent of antibody phage display technology provides a way of producing host-derived mAbs in animals where the molecular genetics of Ig formation are known. Exploiting recent advances in the molecular immunology of cattle, we report here the design of an optimized phage display vector, pComBov, for the construction of combinatorial libraries of bovine Ig antigen-binding fragments (Fab) of native sequence. By using this system, we initially have generated and characterized a panel of bovine mAbs against a model antigen glutathione S-transferase. The isolated mAbs showed features typical of bovine Igs and recognized glutathione S-transferase with high specificity in ELISA and by Western blotting. The pComBov expression system can be readily adapted for the preparation of libraries from related ruminant species and advances the use of monoclonal reagents derived in this way for comparative studies in animals of economic importance.

Amino Acid Sequence↗
PubMed1985

Deletion mapping of human chromosome 5 using chromosome-specific DNA probes.

A complete genomic DNA library was prepared from a Chinese hamster-human cell hybrid that contains human chromosome 5 as its only human DNA. Unique or low-copy DNA fragments, isolated form recombinant bacteriophage that contained human DNA inserts, were regionally mapped on chromosome 5 using Southern blot analysis of genomic DNA from a series of hybrid cell lines that were selected as having deletions of various portions of 5q. The chromosome 5-specific DNA library, together with a genetic selective procedure allowing the isolation of hybrid cell lines with deletions of virtually any portion of 5q, will provide a means to construct very accurate physical and recombinational maps of this human chromosome. This system represents an excellent opportunity to examine very precisely the relationship between physical and genetic distances for many loci along the length of this autosome.

Animals↗
PubMed2002

Characterisation of combinatorial libraries of mucin-2 antigen peptides by high-resolution mass spectrometry.

An epitope motif, TX(1)TX(2)T, of mucin-2 glycoprotein was identified by means of a mucin-2-specific monoclonal antibody, mAb 994, raised against a synthetic mucin-derived 15-mer peptide conjugate. For determination of the epitope sequence recognised with highest affinity by mAb 994, a combinatorial approach was applied using the portioning-mixing technique excluding Cys. Antibody binding of libraries was most profound when Gln was at the X(1) position. Analytical characterisation of the TQTX(2)T library was conducted by amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and electrospray ionisation Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometric methods. Control libraries were prepared by mixing 19 individual peptides corresponding to the TQTX(2)T sequence. Thus, mixtures of 6, 10 and 19 pentapeptides were analysed and compared with the combinatorial mixture. MALDI-TOFMS was able to detect only partially the components in the 6- and 10-member mixtures, but failed to characterise a more complex 19-member mixture. In contrast, ESI-FTICRMS resolved all mixtures of higher complexity and provided direct identification at monoisotopic resolution, such as for a peptide library containing 'isobaric' lysine and glutamine (Delta m = 0.0364 Da). The results of this study suggest that ESI-FTICRMS is a powerful tool for characterisation of combinatorial peptide libraries of higher complexity.

Amino Acid Sequence↗
PubMed2000

A BAC library and paired-PCR approach to mapping and completing the genome sequence of Sulfolobus solfataricus P2.

The original strategy used in the Sulfolobus solfataricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR screening. The PCR approaches included a novel chromosome walking method termed "paired-PCR". 21 gaps were filled by BAC end sequence analyses and 6 gaps were filled by PCR including three large ones by paired-PCR. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BAC clones were selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half the genome sequence has been accumulated.

Chromosomes, Artificial, Bacterial↗
PubMedDate not supplied

Recombinant DNA probes for Mycoplasma synoviae.

A genomic library was prepared from Mycoplasma synoviae (MS) strain WVU 1853 cloned in plasmid vector pUC8 and transformed in Escherichia coli host JM83. In dot blot assays, four transformed E. coli clones hybridized with 32P-labeled chromosomal DNA of MS but not with 32P-labeled chromosomal DNA of M. gallisepticum (MG) strain S6. In Southern hybridization, each of the CsCl-purified recombinant plasmid clones was shown to contain two MS DNA fragments between 1.0 to 2.3 kbp in length. 32P-Labeled probes prepared from each of the four recombinant plasmids hybridized in dot blot assays with MS strain WVU 1853 and nine MS field isolates but not with MG strains S6, K810, F2F10, four MG field isolates, and 15 other species of avian mycoplasmas.

Animals↗
PubMed1996

Detection of potential cytokine gene(s) in an Alu-PCR library from the human chromosome 4.

Jumping and linking libraries have been constructed in order to have more information of chromosome 4. Two types of jumping libraries (Not I and Xma III jumping) and one linking library were prepared. The jumping clones represent DNA regions between two rare-cutting enzymes sites (such is Not I or Xma III) whereas linking clones represent only the area which surrounds the rare-cutting site. These clones were used as target DNA in Alu-PCR. The Alu-PCR fragments were cloned into pGEM-T vector and transformed into Escherichia coli JM 109 competent cells. The colonies were screened for inserts by restriction analysis. More than 50% of the colonies were shown to contain inserts. Inserts from each library were screened by PCR. One of the jumping libraries appeared to have inserts containing CpG islands and AT-rich sequences.

Chromosomes, Human, Pair 4↗
PubMed1989

[Cloning and sequence analysis of salmon prolactin cDNA].

A cDNA library was prepared from pacific Chinook Salmon pituitaries. Salmon prolactin gene was screened using synthetic oligonucleotide probes based on partial protein sequence. A positive clone (PRL-10) was identified and sequenced. It is a full size clone containing 1.1 and coding for a preprolactin of 211 amino acids.

Amino Acid Sequence↗
PubMed2004

Synthesis and biological activities of novel 17-aminogeldanamycin derivatives.

Geldanamycin interferes with the action of heat shock protein 90 (Hsp90) by binding to the N-terminal ATP binding site and inhibiting an essential ATPase activity. In a program directed toward finding potent, water soluble inhibitors of Hsp90, we prepared a library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogs, and compared their affinity for Hsp90, ability to inhibit growth of SKBr3 mammalian cells, and in selected cases, water solubility. Over 20 analogs showed cell growth inhibition potencies similar to that of 17-allylamino-17-demethoxygeldanamycin (17-AAG), the front-runner geldanamycin analog that is currently in multiple clinical trials. Many of these analogs showed water solubility properties that were desirable for formulation. One of the most potent and water-soluble analogs in the series was 17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG), which was independently prepared by the NCI and will soon enter clinical trials. Importantly, the binding affinity of these analogs to the molecular target Hsp90 does not correlate well with their cytotoxicity in SKBr3 cells.

Amino Acid Sequence↗
PubMed1994

Investigation of antigen-antibody interactions using a soluble, non-support-bound synthetic decapeptide library composed of four trillion (4 x 10(12) sequences.

A decapeptide positional-scanning synthetic-peptide combinatorial library (PS-SPCL) made up of four trillion (4 x 10(12) decapeptides was synthesized; its use is illustrated here for the study of a peptide-antibody interaction. This library was prepared by a chemical-mixture approach using a specific ratio of amino acids empirically determined to give approximately equimolar incorporation of each amino acid during each coupling step. Despite the immense number of decapeptides making up each peptide mixture [approx. 200 billion (2 x 10(11)], specific sequences having nanomolar affinities for a peptide-antibody interaction could be readily identified. Upon screening this decapeptide PS-SPCL in this well characterized system, the known six-residue antigenic-determinant sequence was found, with the most specific residues appearing to 'walk through' the ten positions of the peptide library. More importantly, it appears that antibody recognition in this system is stronger when the antigenic determinant is located at the C-terminus of the decapeptide library. Individual decapeptides corresponding to sequences derived from the most active peptide mixtures at each position were synthesized to confirm the results of the screening; 15 peptides were found to have IC50 values between 0.6 and 9.5 nM, four of which were found to be 5-10 times more active than the known six- and 13-residue control peptides. These results further illustrate the power of the positional-scanning peptide library concept, and extend its practical range to a decamer library composed of four trillion (4 x 10(12) decapeptides.

Acetylation↗
PubMed2026

An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

Influenza A virus↗