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 1,117 records · Page 62Linked to original sources
PubMed1998

Structure, diversity, and repertoire of VH families in the Mexican axolotl.

The Mexican axolotl V(H) segments associated with the Igh C mu and C nu isotypes were isolated from anchored PCR libraries prepared from spleen cell cDNA. The eight new V(H) segments found bring the number of V(H) families in the axolotl to 11. Each V(H) had the canonical structural features of vertebrate V(H) segments, including residues important for the correct folding of the Ig domain. The distribution of ser AGC/T (AGY) and TCN codons in axolotl V(H) genes was biased toward AGY in complementarity-determining region-1 (CDR1) and TCN in framework region-1 (FR1); there were no ser residues in the FR2 region. Thus, the axolotl CDR1 region is enriched in DNA sequences forming potential hypermutation hot spots and is flanked by DNA sequences more resistant to point mutation. There was no significant bias toward AGY in CDR2. Southern blotting using family-specific V(H) probes showed restriction fragments from 1 (V(H)9) to 11-19 (V(H)2), and the total number of V(H) genes was 44 to 70, depending on the restriction endonuclease used. The V(H) segments were not randomly used by the H mu and H nu chains; V(H)1, V(H)6, and V(H)11 were underutilized; and the majority of the V(H) segments belonged to the V(H)7, V(H)8, and V(H)9 families. Most of the nine J(H) segments seemed to be randomly used, except J(H)6 and J(H)9, which were found only once in 79 clones.

Ambystoma mexicanum↗
PubMed1998

An antigen recognized by autologous CTLs on a human bladder carcinoma.

By stimulating blood lymphocytes with autologous bladder carcinoma cells that had been transfected with B7-1, we obtained a panel of CTL clones which lyse specifically the bladder tumor cells in an MHC class I-restricted fashion. Based on inhibition with anti-HLA Abs and the recognition of allogeneic tumor cells, we could distribute our clones in three groups that recognized three distinct Ags. We characterized one of these Ags by screening a cDNA library prepared with the RNA from this bladder tumor line. This new tumor Ag is a peptide presented by HLA-B4403 molecules. It is produced by a point mutation in a gene that is recorded in databases under the name KIAA0205, is ubiquitously expressed, and whose function is unknown. We also found this mutation in the tumor sample that was originally resected from this patient, but the mutation was not found in the 100 or more independent tumors of various histologic types that were tested. This report is the first to describe the isolation of CTL clones directed against human bladder cancer and the molecular characterization of a bladder tumor Ag.

Antigens, Neoplasm↗
PubMed1998

Sequencing and chromosomal localization of the RA138 gene encoding a rice allergenic protein.

A cDNA clone (RA138) encoding a rice allergenic (RA) protein has been isolated during a large-scale random sequencing of a cDNA library prepared from developing seeds. The nucleotide sequence of the RA138 gene contained an open reading frame (ORF, 477 bp) encoding a 17 kDa protein. The amino acid sequence deduced from the ORF was composed of 159 amino acid residues and was highly homologous to those from RA genes previously isolated, such as RA5 (92% identity), RA14 (73%), and RA17 (68%). The protein contained 10 cysteine residues that were conserved in the alpha-amylase/trypsin inhibitor family including RA proteins. Excluding a putative signal peptide consisting of 26 amino acid residues, the mature protein would be 14.4 kDa in size and have a pI of 7.0. DNA gel blot analysis under high stringency conditions indicated that multiple copies of the RA138 gene were present in the rice genome. The chromosomal location of the RA138 gene has been identified on chromosome 7 in a segregation analysis using a population of 164 recombinant inbred lines derived from a cross between Milyang 23 and Gihobyeo. The locus that may contain multiple copies of the RA138 was located between RFLP markers RG477A and C492 with genetic distances of 10.7 cM and 6.7 cM, respectively.

Allergens↗
PubMed1998

Two types of mRNA encoding myosin regulatory light chain in carp fast skeletal muscle differ in their 3' non-coding regions and expression patterns following temperature acclimation.

cDNA clones encoding the myosin regulatory light chain (RLC) were isolated from a cDNA library prepared from fast skeletal muscle of the carp Cyprinus carpio L. Two types of cDNA clone encoding carp RLC were found with identical deduced amino acid sequences. The two mRNAs differed in the number of polyadenylation signals prior to the poly(A) tail in the 3' non-coding region. The two mRNA species, with approximate sizes of 1.4 and 0.8 kilobases, were also observed in northern blot analysis. Carp were acclimated for a minimum of 5 weeks to either 10 degreesC or 30 degreesC (14 h:10 h light:dark photoperiod). The total levels of mRNA transcripts coding for the RLC and myosin heavy chain were, respectively, 3.3 and 3.9 times higher in cold- than in warm-acclimated fish. Differences in the levels of RLC in mRNA transcripts were largely due to the concentration of the 1.4 kilobase mRNA species.

Journal Article↗
PubMed1998

Molecular cloning and characterization of a plastid chaperonin 60 alpha subunit from Canavalia lineata.

A 2040 bp cDNA encoding plastid chaperonin 60alpha protein was isolated from a cDNA expression library prepared from Canavalia lineata leaves. The sequence analysis of the clone showed a significant homology to those of other plant plastid chaperonin 60alpha. The genomic DNA blot analysis indicated that Canavalia lineata chaperonin 60alpha is encoded by a single gene in the genome. The short-term kinetics of light on the gene expression revealed that the remarkable accumulation of the gene occurred after 24 h. The mRNA was also detectable in dark-grown seedling leaves.

Amino Acid Sequence↗
PubMed1998

Two types of mRNA encoding myosin regulatory light chain in carp fast skeletal muscle differ in their 3' non-coding regions and expression patterns following temperature acclimation.

cDNA clones encoding the myosin regulatory light chain (RLC) were isolated from a cDNA library prepared from fast skeletal muscle of the carp Cyprinus carpio L. Two types of cDNA clone encoding carp RLC were found with identical deduced amino acid sequences. The two mRNAs differed in the number of polyadenylation signals prior to the poly(A) tail in the 3' non-coding region. The two mRNA species, with approximate sizes of 1.4 and 0.8 kilobases, were also observed in northern blot analysis. Carp were acclimated for a minimum of 5 weeks to either 10 degrees C or 30 degrees C (14h:10h light:dark photoperiod). The total levels of mRNA transcripts coding for the RLC and myosin heavy chain were, respectively, 3.3 and 3.9 times higher in cold-than in warm-acclimated fish. Differences in the levels of RLC in mRNA transcripts were largely due to the concentration of the 1.4 kilobase mRNA species.

Adaptation, Physiological↗
PubMed1998

Strategies for antiviral drug discovery.

The need for antiviral drugs is growing rapidly as more viral diseases are recognized. The methods used to discover antiviral drugs have evolved considerably over the past 40 years and the overall process of discovery can be broken down into subprocesses which include lead generation, lead optimization and lead development. Various methods are now employed to ensure these processes are carried out efficiently. For lead generation, screening methodologies have developed to the extent where hundreds of thousands of compounds can be screened against a particular target. An alternative approach is to use the structures of enzyme substrates as a starting point for drug discovery. Much use is now made of X-ray crystallographic data of target-inhibitor complexes for the optimization of lead structures, and methods for preparing libraries of compounds to assist both generation and optimization of leads are well-developed. The methods used to predict and improve the pharmacokinetic properties of compounds are also changing rapidly. Finally, novel approaches to antiviral therapy using oligonucleotide-based compounds or by modulating the host immune response are also being explored. This review discusses these approaches, provides examples of where their application has been successful and sets them against a historical background.

Anti-HIV Agents↗
PubMed1993

Generation and screening of an oligonucleotide-encoded synthetic peptide library.

We have prepared a library of approximately 10(6) different peptide sequences on small, spherical (10-microns diameter) beads by the combinatorial chemical coupling of both L- and D-amino acid building blocks. To each bead is covalently attached many copies of a single peptide sequence and, additionally, copies of a unique single-stranded oligonucleotide that codes for that peptide sequence. The oligonucleotide tags are synthesized through a parallel combinatorial procedure that effectively records the process by which the encoded peptide sequence is assembled. The collection of beads was screened for binding to a fluorescently labeled anti-peptide antibody using a fluorescence-activated cell sorting instrument. Those beads to which the antibody bound tightly were isolated by fluorescence-activated sorting, and the oligonucleotide identifiers attached to individual sorted beads were amplified by the PCR. Sequences of the amplified DNAs were determined to reveal the identity of peptide sequences that bound to the antibody with high affinity. By combining the capacity for information storage in an oligonucleotide code with the tremendous level of amplification possible through the PCR, we have devised a means for specifying the identity of each member of a vast library of molecules synthesized from both natural and unnatural chemical building blocks. In addition, we have shown that the use of flow cytometry instrumentation permits facile isolation of individual beads that bear high-affinity ligands for biological receptors.

Amino Acid Sequence↗
PubMed1985

A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein.

An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.

Animals↗
PubMedDate not supplied

A new subtraction technique for molecular cloning of rare antiviral antibody specificities from phage display libraries.

The preparation of random combinatorial libraries exposed on the surface of phage provides a route for the selection of diverse high affinity human monoclonal antibodies. However, in particular settings, the isolation of genes coding for a rare antibody can be elusive because some epitopes are predominant and because, in the case of impure antigens, the protein or any compound of interest can be present in relatively minimal amount. In this paper, we describe the successful utilization of a new strategy of "preadsorption" panning that allowed us to clone a rare human monoclonal antibody fragment and to access a different antibody repertoire. The procedure is easy, fast, inexpensive, can be used together with other panning techniques and can be particularly useful in cloning antibodies against rare or unknown determinants.

Antibodies, Monoclonal↗
PubMedDate not supplied

gamma-Hydroxyalkynyl ketones as useful scaffolds for the preparation of combinatorial libraries of furans, isoxazoles and pyrazoles.

Using 5-hydroxy-hex-3-yne-2-one (9) as a model substrate, the utility of the gamma-hydroxyl alkynyl ketone scaffold for the preparation of combinatorial libraries of furans, isoxazoles, and pyrazoles is described. The addition of hydrazoic acid to the acetylenic ketone 9 forms in a single step both 3-azido-2,5-dimethyl furan (10) and alpha,5-dimethyl-3-isoxazolemethanol (11). Reaction of a mixture of (E)- and (Z)-bromoenediones (5 and 6), accessible from 3-bromo-2,5-dimethyl furan (17), with 1,1-dimethylhydrazine afforded a nearly quantitative yield of 1,3-dimethyl-5-acetylpyrazole (18). When the (E)- and (Z)-vinyl bromides 5 and 6 were reacted with sodium azide, 3-acetyl-5-methylisoxazole (7) formed via the intermediate (Z)-3-azido-3-hexene-2,5-dione (4), was the only product.

Journal Article↗
PubMed1990

Differential screening of murine ascites cDNA libraries by means of in vitro transcripts of cell-cycle-phase-specific cDNA and digital image processing.

Cell-cycle-phase-specific cDNA libraries were prepared in the lambda gt10 vector and in the in vitro transcription vector, pBluescript. Plaques of the cDNA libraries prepared in the lambda gt10 vector were differentially screened with (a) in vitro transcripts of the cell-cycle-phase-specific cDNAs cloned in the transcription vector and (b) with first-strand cDNA of mRNA from phase-synchronous cells. The results suggest that first-strand cDNA can be replaced, at least in prescreening experiments, by in vitro transcripts of representative cDNA libraries prepared in in vitro transcription vectors. The fractions of differential clones detected with in vitro transcripts (1.2%) and with first-strand cDNA (1%) were in the same order. Individual clones selected by differential hybridization with in vitro transcripts could be verified by differential hybridization with cell-cycle-phase-specific first-strand cDNA. This indicates that the pattern of stage-specific prevalences of cDNA clones is essentially retained during careful amplifications of large cDNA libraries. The application of in vitro transcripts of stage-specific cDNA for differential screening experiments is of interest in cases where the amount of biological material is either limited or difficult to prepare. It also allows standardization of the probes in repeated screening experiments. Three clones reflecting cell-cycle-phase-specific mRNA prevalences were chosen and analyzed on the sequence level. Two sequences with S-phase prevalences were identified. They code for elongation factor EF1 alpha and for glyceraldehyde-3-phosphate dehydrogenase, respectively. The third sequence reflects the first cDNA of a mRNA with significant prevalence in G2-phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Combinatorial modification of natural products: preparation of unencoded and encoded libraries of Rauwolfia alkaloids.

We report the preparation of combinatorial libraries which consist of derivatives of the stereoisomeric alkaloids yohimbine and rauwolscine-members of the Rauwolfia genus. The chemistry was performed on solid support using the divide-and-pool method, and involved the derivatization of the E-ring carboxylates and hydroxyls of these alkaloids with 36 amino acids and 22 carboxylic acids, respectively, to afford 792 bifunctionalized derivatives. The rauwolscine library was prepared using an encoding strategy in which the identity of each incorporated amino acid was recorded by cosynthesizing chemically inert tags prior to the pooling step. The general strategy for library synthesis exploits existing functionality present on the natural products, and should be applicable to other families of secondary metabolites.

Chromatography, High Pressure Liquid↗
PubMed2005

Direct PCR amplification of various modified DNAs having amino acids: convenient preparation of DNA libraries with high-potential activities for in vitro selection.

We synthesized modified 2'-deoxyuridine triphosphates bearing amino acids at the C5 position and investigated their substrate properties for KOD Dash DNA polymerase during polymerase chain reaction (PCR). PCR using C5-modified dUTP having an amino acyl group (arginyl, histidyl, lysyl, phenylalanyl, tryptophanyl, leucyl, prolyl, glutaminyl, seryl, O-benzyl seryl or threonyl group) gave the corresponding full-length PCR products in good yield. Although dUTP analogues bearing aspartyl, glutamyl or cysteinyl were found to be poor substrates for PCR catalyzed by KOD Dash DNA polymerase, optimization of the reaction conditions resulted in substantial generation of full-length product. In the case of reaction using dUTP analogue having a cysteinyl group, addition of a reducing agent improved the reaction yield. Thus, PCRs using KOD Dash DNA polymerase together with amino acyl dUTP provide convenient and efficient preparation of various modified DNA libraries with potential protein-like activities.

Amino Acids↗
PubMed2001

The multicomponent reactions and their libraries for natural and preparative chemistry.

It was recently recognized that three different types of multi-component reactions (MCRs) exist. In preparative chemistry, the MCRs of type II form their products particularly efficiently. These reactions correspond to equilibria of educts and intermediate products, whose final products are formed practically irreversibly. In recent years, the four component reaction of the isocyanides (U-4CR) of type II and their unions with various reactions and MCRs have become an important industrial process for preparing products and their libraries. It has been demonstrated that all conceivable collections of U-4CR educts can be converted into the corresponding products. In the usual chemical reactions, only the substituents of the products can be varied, whereas the U-4CR and related reactions can also produce skeletally different types of products with diverse substituents. The preparative advantages of forming products by the one-pot MCRs and the great variety of the possible products are illustrated in this review.

Combinatorial Chemistry Techniques↗
PubMed1995

An arrayed library enriched in hncDNA corresponding to transcribed sequences of human chromosome 19: preparation and analysis.

A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains around 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs.

Animals↗
PubMedDate not supplied

Preparation of a recombinant cDNA library from poly(A+) RNA of the Alzheimer brain. Identification and characterization of a cDNA copy encoding a glial-specific protein.

Studies were undertaken to assess the extent to which messenger RNA prepared from the postmortem Alzheimer's disease (AD) brain can be used for the successful preparation of a recombinant cDNA library. Initial experiments focused on the glial-specific marker glial fibrillary acidic protein (GFAP) since GFAP expression appeared to be a model for further studies on mRNAs that may continue to be expressed at high levels in the vicinity of lesioned sites in the AD brain. An AD cDNA library, prepared in the lambda gt11 expression vector system contained GFAP-specific recombinants. One of these was sequenced and the insert was shown to exhibit 88% homology with the similar sequence from mouse GFAP. As established by Northern blots, the size of the GFAP mRNA prepared from the routinely acquired postmortem AD cortex, approximately 2.7 kb, was the same as from a neurologically normal control brain. These results agree with earlier studies on GFAP mRNA from fresh mouse brain. The results demonstrate that in the postmortem AD brain, astroglial-specific mRNA remains sufficiently stable for molecular genetic analysis and may serve as a useful model for examining the genetic expression of mRNAs that may be related to the molecular pathogenesis and the etiology of AD.

Aged↗