Showing papers in "Methods in 1991"

TL;DR: A brief evaluation of commercial efforts to automate DNA sample preparation, separation, and data acquisition is presented, including instruments currently available as well as new concepts potentially amenable to automation.

TL;DR: This method provides the most expeditious route to monoclonal antibodies yet described and is described as a revolutionary method for the rapid selection of monoconal antibodies.

TL;DR: Results of searches performed using BLASTN's default score matrix are compared with those using scores based on a mutational model in which transitions are more prevalent than transversions.

TL;DR: Efficient assembly of sFv genes by polymerase chain reaction, oligonucleotide-directed mutagenesis, and synthetic genetic methods is facilitated by the conserved signal, constant, framework, and linker segments that flank the variable domains.

TL;DR: A technique for rapidly searching libraries of protein variants for ones that bind tightly to a target receptor is described, and several libraries containing >10 6 variants of human growth hormone are sorted for those that bind with greater affinity to the extracellular domain of the human growth hormones receptor.

TL;DR: Linear amplification sequencing is a simple, general-purpose sequencing method using Taq polymerase in cycled rounds of primer extension in the presence of deoxynucleotides and dideoxyn nucleotides that should prove useful in large-scale seuencing projects using automatic sequencing machines as well as in the sequence analysis of individual clones.

TL;DR: General considerations for selection of appropriate combinations of fluorescent dyes for immunofluorescence, conjugation of fluorochromes with protein probes, and quantitation of fluorescence are reviewed.

TL;DR: A set of vectors that use a powerful cytomegalovirus promoter enhancer to direct expression of the coding sequence for each immunoglobulin chain is described, allowing the generation of cell lines that can accumulate in excess of 200 mg/liter of antibody in the culture medium.

TL;DR: The optimum conditions for extraction of pigment-proteins from photosynthetic membranes with glycosidic surfactants and their subsequent fractionation by nondenaturing PAGE to purify them in a state as close as is currently possible to their native state are described.

TL;DR: The method makes it possible to construct antibody libraries in phage without resort to hybridoma technology and sensitive studies of B-cell differentiation and ontogeny, B-lymphoma oncogenesis and heterogeneity, and antibody diversity, as well as immortalization and manipulation of the antibody repertoire for diagnostic and therapeutic purposes.

TL;DR: Detailed methods for the isolation, lysis, amplification, and detection of alleles in single cell samples are given, with an emphasis on protocols for individual sperm.

TL;DR: It is suggested that the efficiency of random DNA sequencing may be improved by simple modifications facilitating closure, and strategies for overlapping contigs and sequencing gaps are suggested and discussed in detail.

TL;DR: A general method for the high-level expression of authentic antibodies in a eukaryotic host is described and the antibodies recovered from the supernatants of infected cultures behaved indistinguishably from B-cell-derived immunoglobulins with similar specificities.

TL;DR: Methods for the in vitro selection and amplification of rare functional nucleic acid molecules are described, including strategies and applications for pool construction, selection, and amplification.

TL;DR: A rapid route for the generation of monoclonal antibodies by repertoire cloning is described, which circumvents the need for conventional hybridoma production and allows a variety of new approaches in antibody design and selection.

TL;DR: The use of two-dimensional neutral-alkaline and neutral-chloroquine gels to study normal and aberrant simian virus 40 DNA replication intermediates and covalent or topological linkages between parental and daughter DNA strands is described.

TL;DR: A sequencing strategy that combines these isolation and sequencing methods with initial shotgun cloning followed by a short oligonucleotide primer walking approach for both contig joining and proofreading is described, which significantly increase the rate and ease of DNA sequence data acquisition, as well as improve the extent and accuracy of the sequence data.

TL;DR: Of the various single-sided PCR strategies, ligation-mediated PCR is uniquely suited to the amplification of a genomic sequence ladder because it preserves the single base resolution present in the starting material.

TL;DR: A detailed explanation of the FACS-Gal assay, used to measure the intracellular concentration of β -galactosidase, as expressed by an introduced Escherichia coli lacZ gene, and two examples of how it has been used to answer questions that are unapproachable with standard reporter gene systems.

TL;DR: Degenerate PCR primers that target regions of highly conserved amino acid sequence that are characteristic of a given family will selectively amplify members within that multigene family.

TL;DR: Several refinements of O'Farrell's original technique that lead to improved reproducibility, resolution, and detection sensitivity are discussed, and the advantages and versatility of a mid-sized gel format are emphasized.

TL;DR: Two techniques for adding an anchor to the unknown gene segments and amplifying with the anchor and a specific primer are described, the first in which an anchor is added to cDNA by terminal deoxynucleotidyl transferase, and the second in which a anchor is ligated onto double-stranded DNA.

TL;DR: Methods for the production and use of human DNA fragments using the polymerase chain reaction and oligonucleotide primers directed to repetitive sequences are described and interpretation of patterns of amplification in somatic cell hybrids by ethidium bromide staining and hybridization analysis is discussed.

TL;DR: Important attributes are: (i) GMDs are physically manipulable and can be handled much like cells (e.g., suspended, pipetted, centrifuged), and (ii)GMDs rapidly exchange molecules with the external medium by diffusion, which allows rapid changes in the exposure of individual cells and microcolonies within GMDs to many different chemical conditions.

TL;DR: Quantitative analysis is relatively new but the same in every respect except that reagents for staining must be used under saturating conditions and controls must be carefully designed.

TL;DR: In this paper, the techniques for the expression of various antibody fragments in Escherichia coli were summarized and several strategies for expression were compared, focusing on the secretory approach, as it leads directly to functional fragments and thus forms the basis for all screening approaches, whether with cells or phages.

TL;DR: A detailed protocol for two-dimensional DNA typing of mammalian (human and cattle) and plant (tomato) genomic DNA using micro- and minisatellite core probes and spot patterns comprising 100–800 spots are described.

TL;DR: Antibody repertoires can be created following the insertion into transgenic mice of either rearranged or germline configuration immunoglobulin gene DNA, leading, for example, to the production of a human antibody repertoire intransgenic mice.

TL;DR: Two methods in which the polymerase chain reaction (PCR) is used for site-specific mutagenesis and for DNA recombination without any enzymatic reaction in vitro apart from DNA amplification are described.

TL;DR: Of the methods available for the construction of chimeric genes, the linker-adapter approach is the simplest and often the most rapid technique; however, in practice this method is not always feasible.