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PubMed2006

Developing and sustaining a web-based library newsletter.

The JEFFLINE Forum, Scott Library's attempt to revive the traditional library newsletter by employing the technical advantages and increasing presence of the Web, debuted in October 1999. The fifth anniversary of the Forum was a good time to look back at its evolution and to examine some of the challenges inherent in developing and sustaining a library newsletter. By identifying the most successful strategies, and reflecting on the lessons learned along way, the authors hope to continue the evolution of the Forum and help to ensure the viability of the library newsletter.

Humans↗
PubMed2007

Integrating and promoting medical podcasts into the library collection.

Podcasts offer a way for medical professionals to listen to current information in medicine from an iPod, MP3 player, handheld device, or computer. As podcasts become more popular, libraries may be interested in integrating programs into the library collection. South Pointe Medical Library evaluated medical podcasts relevant to the scope of the library collection and explored methods for finding and organizing such programs in ways that are efficient for both the librarian and the patron. doi:10.1300/J115v26n01_03.

Access to Information↗
PubMed2003

An improved method for plant BAC library construction.

Large genomic DNA insert-containing libraries are required as critical tools for physical mapping, positional cloning, and genome sequencing of complex genomes. The bacterial artificial chromosome (BAC) cloning system has become a dominant system over others to clone large genomic DNA inserts. As the costs of positional cloning, physical mapping, and genome sequencing continuously decrease, there is an increasing demand for high-quality deep-coverage large insert BAC libraries. In our laboratory, we have constructed many high-quality deep-coverage large insert BAC libraries including arabidopsis, manocot and dicot crop plants, and plant pathogens. Here, we present the protocol used in our laboratory to construct BAC libraries.

Chromosome Mapping↗
PubMed2004

Protein arrays from cDNA expression libraries.

This chapter describes the production of a cDNA expression library from human fetal brain, the construction of a high-density protein array from such a library, and two applications to screen the array for binding proteins. After producing the library and decollating the expression clones, one can pick thousands of expression clones with a laboratory robot and can deposit them into microtiter plates in an ordered manner. Such ordered clone libraries are the starting material for the construction of a high-density protein array. This array is constructed by spotting the expression clones onto a protein-binding membrane. Following cell growth and induction of protein expression on the membrane, the cell spots are lysed and their recombinant protein immobilized on the membrane. The so-constructed array carries thousands of proteins without the need to clone, express, and spot individual proteins. Such arrays allow one to screen for numerous protein functions in a high-throughput manner.

Amino Acid Sequence↗
PubMed2002

Adrenergic receptor genes: cDNA and genomic library construction.

Adrenergic receptors mediate the central and peripheral actions of norepinephrine and epinephrine and are pharmacologically divided into three major types, alpha-1, alpha-2, and beta. These types are further subdivided into alpha-1A, alpha-1B, and alpha-1D; alpha-2A, alpha-2B, and alpha-2C; and beta-1, beta-2, and beta-3, respectively. Adrenergic receptor sequence information is presented in three tables with respect to species, subtype identification, GenBank accession number, source of the nucleic acid sequence, the presence of a 5' flanking region upstream of the transcription start site, and the nucleotides defined as introns, coding regions, or 3' and/or 5' untranslated but transcribed (UTR) regions. Sequences have been assigned to adrenergic subtype categories based on sequence comparison using either FASTA or denogram of Pileup from the GCG sequence analysis program rather than as described in the author definition line. Sequence information found in these tables can be important for probe development for screening libraries for isolating adrenergic receptor genes from species other than the most common species. Where commercial libraries for specific tissue or species needs are not available, we have described construction of genomic cosmid libraries or PCR-based synthesis of a cDNA library using a microgram of RNA.

Animals↗
PubMed2004

Normal/disease-paired cDNA library subtraction for molecular marker development.

This study consists of a novel strategy for the identification of potential cancer exclusively expressed genes, which might lead to the development of valuable diagnostic molecular markers. Normal- and cancer-paired tissues from the same patient were collected and subjected to the construction of primary complementary deoxyribonucleic acid (cDNA) libraries. The cancer cDNA library was generated by subtracting normal cDNAs from the primary cancer library. The remaining clones in the subtracted cancer library were sequenced and Basic Local Alignment Search Tool (BLAST)-analyzed against current nonredundant and est_human databases in the GenBank. The clones that had no matches with any known gene sequences except the human genomic sequences were identified as ideal candidates for potential molecular marker development. The candidate marker sequence and associated primer sequences were identified on the target clone sequence of the region with the least, or no, matched homologs. The specificity of the markers was measured by a polymerase chain reaction test experiment with the DNA templates from different human normal tissues, genomic DNA, and additional patients' tissues. Results from bioinformatical and experimental approaches used suggest that the methodology has a high potential to identify cancer exclusive transcripts. Thus, it might result in the development of diagnostic markers.

Biotechnology↗
PubMed2006

Mutant library construction in directed molecular evolution: casting a wider net.

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).

Animals↗
PubMedDate not supplied

Towards the physical map of the Trypanosoma cruzi nuclear genome: construction of YAC and BAC libraries of the reference clone T. cruzi CL-Brener.

Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease.

Animals↗
PubMed2001

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics.

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.

DNA, Complementary↗
PubMed2001

Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing.

We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length normalized cDNA libraries, of which the average residual polyA tail was 4 bases or shorter in most of the clones analyzed Because of the removal of homopolymeric stretches, libraries prepared with this method can be used for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.

Animals↗
PubMed2002

Generation of von Willebrand factor epitope libraries expressed in E. coli.

The von Willebrand factor (VWF) subunit is composed of several domains, often coinciding with structural regions, characterized through their specific interaction with a ligand. Since several monoclonal antibodies (MoAbs) have been shown to functionally interfere with one of the specific interactions, we have created libraries of bacterial clones expressing peptidic sequences of VWF to map antibodies directed against this protein. Randomly cleaved fragments of VWF cDNA have been cloned in a plasmid designed for the expression of small peptides as part of larger fusion proteins. The NovaTope system is a useful procedure for protein analysis, allowing screening of epitopes composed of contiguous amino acid residues. To map MoAbs with conformational discontinuous epitopes displayed on small as well as large peptidic domains, this technique had to be widely modified to obtain two VWF peptide libraries expressing two ranges of peptide length (15-70 and 100-300 amino acids). Screening with six MoAbs with an epitope in a known region was performed to control both libraries. Four MoAbs were mapped through the characterization of overlapping sequences for 5-10 different positively expressed clones respectively. Two of these mapped MoAbs had no known inhibitory effect and bind reduced VWF only. The fact that the two other MoAbs mapped VWF functional interactions with ligands, platelet GPIIb/IIIa and Factor VIII, respectively, demonstrate that our libraries are valuable tools to determine conformational epitopes.

Antibodies, Monoclonal↗
PubMed2002

Use of degenerate primers and touchdown PCR for construction of cDNA libraries.

Optimized construction of low-redundancy cDNA mini-libraries using low-stringency RT-PCR is described cDNAs are generated using arbitrary consensus-degenerate hybrid oligonucleotide primers and nanogram amounts of Schistosoma mansoni mRNA. A number of conditions such as temperature and salt concentration are combined to create balanced, low-stringency conditions that permit a normalized amplification of a diversified, random set of sequences. On average, 350 different sequences are obtained per mini-library, which represents a significantly higher diversity of messages per library when compared to previously published conditions (ie., 20-40 sequences/ mini-library). The optimized high-throughput approach described here is likely to help in the discovery of expressed genes in any complex organism.

Animals↗
PubMed2003

Computational methods for sequence mapping of large combinatorial libraries and deduced sequence signatures.

Here we describe a computational approach for the high-throughput sequence mapping of combinatorial libraries obtained by DNA shuffling. Original algorithms and their software implementation were developed for the automated and reliable analysis of hybridization data of differentially labeled oligonucleotide probes with PCR products spotted on DNA microarrays. This novel approach allows a context-dependent sequence attribution tolerant to fluctuations in experimental conditions and is well adapted to hybridization signals of variable qualities resulting from high-throughput PCR amplification from colonies. In addition, the analysis permits the calculation of sequence signatures that are characteristic of combinatorial library structure, defects, and diversity. The approach is of interest for the characterization and the equalization (library reduction to nonredundant structures) of combinatorial libraries involved in directed evolution and could be extrapolated to high-throughput polymorphism analysis.

Biotechnology↗
PubMed2004

High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis.

Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.

DNA, Plant↗
PubMed1996

Mapping the Trypanosoma cruzi genome: analyses of representative cosmid libraries.

In order to generate contiguous cosmid coverage of the genome of the protozoan parasite Trypanosoma cruzi for large-scale sequence analysis, a cosmid library of 36864 individual, primary clones was generated. Total genomic DNA of the reference strain CL Brener was fragmented both by partial digestion with MboI and by physical shearing. For cloning, a modified cosmid vector was used that simplifies analyses such as restriction mapping. The library's representation is about 25 genome equivalents, assuming a size of 55 Mb per haploid genome. No chimerism of inserts in the clones could be detected. The colinearity between cosmid inserts and genomic DNA was verified. Also, hybridizations to the gel-separated karyotype of the organism were carried out as a quality check. Gridded onto two nylon filters, the library was analyzed with a variety of probes. Apart from being used for combined physical and transcriptional mapping of the genome, library filters and clones are also available to interested parties.

Animals↗
PubMed2002

Optimisation of a peptide library for screening specific RNA ligands by flow-injection NMR.

Flow-injection NMR is a very attractive technique for identifying weak but specific ligand-macromolecule interactions. The low-sensitivity of this method is, however, a major concern which hinders its widespread application to large-scale screening. Focussing on RNA-targeting ligands, we have shown in this report that it is possible to design a peptide library that will simultaneously satisfy a number of physico-chemical restraints and sample the sequence space in an "optimal" fashion. This library, which contains less than 200 peptides, covers most of the allowable tripeptide sequences in a non-redundant fashion. In addition, for almost any allowable tetrapeptide sequences, the library contains a close neighbor that will differ at the most by a single conservative replacement. A subset of this library was actually synthesised and used in a preliminary screen against human tRNA(Lys)(3), the primer of HIV-1 reverse transcriptase.

Algorithms↗
PubMed2002

Probing the phosphopeptide specificities of protein tyrosine phosphatases, SH2 and PTB domains with combinatorial library methods.

Protein tyrosine phosphatases, SH2 and PTB domains are crucial elements for cellular signal transduction and regulation. Much effort has been directed towards elucidating their specificity in the past decade using a variety of approaches. Combinatorial library methods have contributed significantly to the understanding of substrate and ligand specificity of phosphoprotein recognizing domains. This review gives a brief overview of the structural characteristics of protein tyrosine phosphatases, SH2 and PTB domains and their binding to phosphopeptides. The chemical synthesis of peptides containing phosphotyrosine or phosphotyrosine mimics and the various formats of synthesis and deconvolution of combinatorial libraries are explained in detail. Examples are given as how different combinatorial libraries have been used to study the interaction of phosphopeptides with SH2 domains and phosphatases. The intrinsic advantages and difficulties of library synthesis, screening and deconvolution are pointed out. Finally, some experimental results on the substrate specificity of protein tyrosine phosphatase 1B and the SH2 domain of the adaptor protein Grb-2 are summarized and discussed.

Binding Sites↗
PubMed2004

Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential.

AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential. METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5alpha competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing. RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13, ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes. CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.

Animals↗