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At least 1,153 records · Page 64Linked to original sources
PubMedDate not supplied

[A method for the preparation of normalized cDNA libraries enriched with full-length sequences].

We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.

Animals↗
PubMed1993

Identification and cloning of a new category of DNA fragments which are poorly represented in human genomic libraries.

We have developed an alternative strategy for the preparation of genomic libraries that ensures better representation of genomic sequences commonly underrepresented in genomic libraries constructed using standard protocols. To overcome the apparent bias against genomic sequences containing clusters of restriction sites we have used nonoptimized restriction digestions to generate a mixture of DNA fragments which have been cloned into the EMBL3 vector. To validate this protocol we have screened the EMBL3 library to identify a full length genomic clone of the prolactin-inducible gene (PIP). Screening 4 other, commercially available, genomic libraries prepared using standard protocols for restriction digestion of the genomic DNA failed to identify any full length clones. We show that this increase in the representation of the full length PIP gene in the EMBL3 genomic library is attributable to the method of insert preparation used and suggests that an additional subset of sequences that may be poorly represented in, or absent from, established libraries may be cloned using this modified protocol.

Base Sequence↗
PubMedDate not supplied

[Gene library of Dunaliella salina, prepared in a cosmid vector of new design].

The gene library (56,160 clones) of the halotolerant alga Dunaliella salina was obtained using a novel cosmid vector pBbv-cosII. The mean size of the insertions is 35-45 kb. Hybridization screening demonstrated that both nuclear and chloroplast DNA-containing clones are presented in the library.

Cell Nucleus↗
PubMed2000

Analysis of libraries encoded with GC tags: compound elution, tag decode analysis, and statistical sampling analysis.

Libraries encoded with electrophoric tags present a unique challenge with respect to library quality control and characterization. Libraries are prepared on Tentagel resin in 200-fold redundancy wherein each resin particle contains one compound per one tag set. The amount of compound present on the bead is ca. 200-500 pmole while tag levels are estimated at 0.5-1 pmol/bead. Several quality control protocols have been developed in order to accurately estimate bead yield and purity for the entire library, ensure high tag fidelity, and to determine the overall performance of individual synthons. This review provides a unique, collective portrait of Pharmacopeia's approach in assessing the quality of libraries prepared using its molecular encoding technology.

Chromatography, Liquid↗
PubMed2003

Preparation of a whole genome phage library using fragmented Escherichia coli genome and its characterization of protein binding properties by surface plasmon resonance.

A novel phage library has been prepared using the Escherichia coli genome digested with three restriction enzymes. The resulting DNA fragments were ligated to the expression vector pCANTAB5 to obtain the library of recombinant M13 phages displaying relatively long exogenous peptides. The library was screened to isolate recombinant phages with high affinity to alkaline phosphatase (AP) from calf intestine. After four rounds of panning three phages (AP1, AP2 and AP3) were shown to have specific binding properties toward AP by enzyme-linked immunosorbent assay. The phages were further characterized by surface plasmon resonance (SPR). Among the three phages AP3 bound the AP-immobilized sensor chip most and caused the highest resonant angle shift. The sensor response decreased with the decrease of the concentration of AP3 added. Furthermore, displacement of AP3 from the AP-immobilized sensor chip was observed upon injection of AP solution to the SPR system, whereas injection of bovine serum albumin solution led to the great increase of the sensor response. This result indicates the specific binding of AP3 to AP.

Alkaline Phosphatase↗
PubMed2001

Identification and initial characterization of 5000 expressed sequenced tags (ESTs) each from adult human normal and osteoarthritic cartilage cDNA libraries.

OBJECTIVE: To prepare, sequence and analyse adult human cartilage cDNA libraries to study the gene expression pattern between normal and osteoarthritic cartilage. METHODS: Poly A(+)RNA from adult human normal and osteoarthritic articular cartilage was isolated and used to prepare cDNA libraries. Approximately 5000 ESTs from each library were sequenced and analysed using bioinformatic tools. The expression of select genes was confirmed by Northern blot and in situ hybridization analysis. RESULTS: Multiple gene families including several classical cartilage matrix protein encoding genes were identified. Approximately 28-40% of the genes sequenced from these libraries were novel, while half of the genes encoded known proteins and 4-6% of the genes encoded novel homologs of known proteins. Several known genes, whose expression has not been reported previously in cartilage, were also identified. We have confirmed the cartilage expression of three known (CTGF, CTGF-L and clusterin) and two novel homologs of known genes (PCPE-2 and Gal-Nac transferase) by Northern blot and in situ hybridization analysis. CONCLUSION: This is the first report of the preparation and sequencing of cDNA libraries from adult human normal and osteoarthritic articular cartilage. Further analysis of genes identified from these libraries may provide molecular targets for diagnosis and/or treatment of osteoarthritis (OA).

Adult↗
PubMed2002

[Mycolic acids analysis from various species mycobacterium by high pressure liquid chromatography (HPLC)].

HPLC is the most useful method to analyze various species of mycobacteria by using mycolic acids. The purpose was to prepare a library containing chromatographic patterns of mycolic acids derived from reference species Mycobacterium, which had been cultivated in standard conditions. 28 reference strains (27 ones from American Type Culture Collection and one cultivated from the vaccine M. bovis BCG) were used. The analysis of mycolic acids involved chromatographic separation of their bromophenacyl derivatives according to Centers for Disease Control recommendation. Mycolic acids profiles formed by HLPC were reproducible for all reference species in this study. Standard deviation of relative retention time of every peak did not exceed 2.5%. The species included into M. tuberculosis complex beyond M. bovis BCG shared the same mycolic acids pattern. HPLC is the only mean to distinguish M. tuberculosis from M. bovis BCG. The other studied stains had species specific patterns which differed from M. tuberculosis complex and M. bovis BCG. The prepared library comprising 28 reference elution profiles of mycolic acids from known mycobacteria species can be applied in diagnostic procedure of tuberculosis and mycobacteriosis.

Chromatography, High Pressure Liquid↗
PubMed1995

Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries.

The discovery of useful peptide substrates for proteases that recognize many amino acids in their active sites is often a slow process due to the lack of initial substrate data and the expense of analyzing large numbers of peptide substrates. To overcome these obstacles, we have made use of bacteriophage peptide display libraries. We prepared a random hexamer library in the fd-derived vector fAFF-1 and included a "tether" sequence that could be recognized by monoclonal antibodies. We chose the matrix metalloproteinases stromelysin and matrilysin as the targets for our studies, as they are known to require at least 6 amino acids in a peptide substrate for cleavage. The phage library was treated in solution with protease and cleaved phage separated from uncleaved phage using a mixture of tether-binding monoclonal antibodies and Protein A-bearing cells followed by precipitation. Clones were screened by the use of a rapid screening assay that identified phage encoding peptide sequences susceptible to cleavage by the enzymes. The nucleotide sequence of the random hexamer region of 43 such clones was determined for stromelysin and 23 for matrilysin. Synthetic peptides were prepared whose sequences were based on some of the positive clones, as well as consensus sequences built from the positive clones. Many of the peptides have kcat/KM values as good or better than those of previously reported substrates, and in fact, we were able to produce stromelysin and matrilysin substrates that are both the most active and smallest reported to date. In addition, the phage data predicted selectivity in the P2 and P'1 positions of the two enzymes that were supported by the kinetic analysis of the peptides. This work demonstrates that the phage selection techniques enable the rapid identification of highly active and selective protease substrates without making any a priori assumptions about the specificity or the "physiological substrate" of the protease under study.

Amino Acid Sequence↗
PubMed1997

Phosphorus-based combinatorial libraries: use of amino acid derivatives as synthons.

Phosphorus has been used as a scaffold to prepare combinatorial libraries of phosphoramidates in which one of the diversity elements resulted from derivatives of amino acids. A small library was prepared for analytical and characterization purposes, followed by a larger library of approximately 8800 compounds. Libraries were assembled on solid supports using the conventional pool-and-divide method, followed by cleavage from the supports at the end of the synthesis. Mass spectrometry was used to confirm that library synthesis had been successful. Individual compounds were also prepared to study the stability of compounds of this type.

Amino Acids↗
PubMed1998

A simple, inexpensive apparatus for performance of preparative scale solution phase multiple parallel synthesis of drug analogs. I. Preparation of a retrospective library of quinolone antiinfective agents.

A simple inexpensive apparatus is described consisting of conveniently commercially available components which is suitable for the solution phase multiple parallel synthesis of 24-72 analogs of drug-like molecules. The use of the apparatus is illustrated by preparation of a retrospective library of over 100 analogs of antimicrobial fluoroquinolones prepared in 0% to quantitative yields. Each analog was prepared in up to 150 mg quantity and each was analyzed by NMR and mass spectrometric techniques to verify its purity and identity.

Anti-Infective Agents↗
PubMed1988

Isolation and characterization of a recombinant lambda gt11 bacteriophage which expresses an immunoreactive Mycobacterium intracellulare protein in Escherichia coli.

Disseminated Mycobacterium avium-Mycobacterium intracellulare (M. avium complex) disease is a prevalent opportunistic infection in patients with acquired immune deficiency syndrome. Because of the increasing importance of this disease, an M. avium complex lambda gt11 expression library was prepared. We screened the library with an absorbed anti-M. intracellulare serum and identified a recombinant phage which expressed a 190-kilodalton beta-galactosidase-M. intracellulare fusion protein. Lysates containing the 190-kilodalton fusion protein evoked strong humoral and cell-mediated responses. The immunoreactivity of the M. intracellulare recombinant protein suggests that antigens isolated from the expression library may be useful as skin test, serodiagnostic, or immunoprophylactic reagents for M. avium complex disease.

Animals↗
PubMed1995

Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue constituents of the gastrulating mouse embryo.

A total of 5 conventional, directionally cloned plasmid cDNA libraries have been constructed from the entire embryonic region of the mid-gastrulation mouse embryo and from its four principal tissue constituents (ectoderm, mesoderm, endoderm and primitive streak). These libraries have been validated with respect to the number of independent clones, insert-size and appropriate representation of diagnostic marker genes. Subtractive hybridisation has been used to remove clones common to the Endoderm and Mesoderm cDNA libraries resulting in an Endoderm minus Mesoderm subtracted library. Probe prepared from this subtracted library has been hybridised to a grid containing approximately 18,500 Embryonic Region library clones. Three novel clones have been recovered as well as expected genes already known to be highly expressed in the primitive endoderm lineage at this stage of development. In situ hybridisation to early postimplantation embryos has revealed the expression patterns of these novel genes. One is highly expressed exclusively in visceral endoderm, one is expressed in ectodermal and endodermal tissues, and the third proves to be an early marker of prospective and differentiated surface ectoderm as well as being expressed in endoderm and its derivatives.

Animals↗
PubMed2005

Preparation of cherry-picked combinatorial libraries by string synthesis.

String synthesis [1-3] is an efficient and cheap manual method for preparation of combinatorial libraries by using macroscopic solid support units. Sorting the units between two synthetic steps is an important operation of the procedure. The software developed to guide sorting can be used only when complete combinatorial libraries are prepared. Since very often only selected components of the full libraries are needed, new software was constructed that guides sorting in preparation of non-complete combinatorial libraries. Application of the software is described in details.

Combinatorial Chemistry Techniques↗
PubMed1972

The preparation and use of library manpower.

Trends in library education and manpower utilization in general are briefly surveyed, with special reference to the official ALA Policy on Library Education and Manpower approved in July 1970.The trends are marked by general upgrading of standards and content of library education, resulting in a longer period of formal education with more room for intensive specialization; different kinds of preparation for different levels of responsibility; and a serious exploration of the ways in which paraprofessional or supportive staff can be utilized to release the librarian for more professional responsibilities.The Policy Statement is analyzed to show its support of most of these developments. A key concept is that the demands of the position should be the test of professional quality, and not the diploma held by the applicant. The hope is that the changes suggested will improve the quality of library service and the preparation for it and that this general improvement will be applicable also to such special fields as medical librarianship.

Journal Article↗
PubMed2005

Diverted total synthesis: preparation of a focused library of latrunculin analogues and evaluation of their actin-binding properties.

Two largely catalysis-based and highly convergent total syntheses of latrunculin A (1) and B (2) were diverted to the preparation of a focused library of analogues of these potent actin-binding macrolides that enjoy widespread use in chemical biology. Because the chosen route allows for structural variations of all characteristic parts of the natural leads, it was possible to map the previously largely unknown structure/activity profile of this class of bioactive natural products. This led to the discovery that the removal of the methyl branches decorating the macrocycle in 2 engenders a significant increase in potency, while streamlining the synthesis to a considerable extent. Moreover, compelling evidence is provided that the conspicuous 2-thiazolidinone ring present in all naturally occurring latrunculins may be an optimal but not an essential structural motif for actin binding because it can be replaced by an oxazolidinone moiety with only slight loss in efficacy. Likewise, the inversion of the absolute configuration of the chiral center at C.16 is well accommodated. From the purely chemical perspective, this investigation attests to the maturity of alkyne metathesis, a method that has received attention as efficient means for the formation of macrocycles only recently.

Actin Cytoskeleton↗
PubMedDate not supplied

The use of high-throughput synthesis and purification in the preparation of a directed library of adrenergic agents.

A library of potential agonists and antagonists for adrenergic receptors was prepared using high-throughput solution-phase parallel synthesis. Traditional solution-phase reductive amination reactions followed by rapid purification by ion exchange chromatography yielded products with near-analytical purity. An array of ketones and amines, arranged in an 8 x 12 matrix, were combined to form 96 individual compounds.

Adrenergic Agonists↗
PubMed1985

[Construction of a gene library using partial filling of DNA sticky ends].

To prepare gene libraries, the incomplete filling of protruding ends has been used. DNAs from phages EMBL 3 and EMBL 3a were sequentially digested with SalI and EcoRI, followed by addition of dTTP, dCTP, and DNA polymerase I (Klenow's fragment). Separately, a genomic DNA was partially cleaved with Sau3AI, followed by addition of dATP, dGTP, and Klenow's fragment. The fragmented phage and genomic DNAs were mixed and ligated, and the recombinant DNAs packed in vitro with the phage proteins. The effectiveness of packaging per microgram of genomic DNA was 10(5) to 10(6) (for the wild phage DNA, 10(7)). The proposed procedure is very rapid and needs only microgram quantities of genomic DNA for preparing a representative gene library. It is also useful for other vectors, containing SalI sites.

Animals↗
PubMed2000

Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes.

In the effort to prepare the mouse full-length cDNA encyclopedia, we previously developed several techniques to prepare and select full-length cDNAs. To increase the number of different cDNAs, we introduce here a strategy to prepare normalized and subtracted cDNA libraries in a single step. The method is based on hybridization of the first-strand, full-length cDNA with several RNA drivers, including starting mRNA as the normalizing driver and run-off transcripts from minilibraries containing highly expressed genes, rearrayed clones, and previously sequenced cDNAs as subtracting drivers. Our method keeps the proportion of full-length cDNAs in the subtracted/normalized library high. Moreover, our method dramatically enhances the discovery of new genes as compared to results obtained by using standard, full-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.

Animals↗