Biomedical subjects

T Hultman

Publications and source records attributed to T Hultman.

11 recordsLinked to original sources

PubMed1992

T cell receptor diversity and activation markers in the V delta 1 subset of rheumatoid synovial fluid and peripheral blood T lymphocytes.

In the present study we have characterized the gamma/delta T cell receptor (TcR) population in synovial fluid (SF) and peripheral blood (PB) of patients with chronic inflammatory arthritis. By double staining we have shown that (a) synovial V delta 1+ cells have a high expression of activation markers CD45R0 ("memory cells") and HLA-DR as compared to PB, indicating a preactivated population of V delta 1-carrying T cells in vivo and (b) interleukin 2-induced expansion of synovial cells yields a high proportion of gamma/delta in most samples expressing predominantly the V delta 1 TcR. Junctional sequence analysis of the TcR delta chain from interleukin 2-expanded PB cell lines demonstrated a polyclonal V delta 1 population in three out of three samples. In SF cell lines three out of four samples were polyclonally expanded. In SF from one patient, however, a limited repertoire of expressed V delta 1 genes was found. Altogether, our data demonstrate the presence of preactivated V delta 1-expressing cells in the synovial compartment. This V delta 1 population is predominantly polyclonal, except in one patient where oligoclonally expanded V delta 1 cells were detected.

Antigens, CD↗

PubMed1992

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.

Automation↗

PubMed1991

Genetic analysis of the polymorphism of the human apolipoprotein E using automated solid-phase sequencing.

A direct sequencing approach has been used to analyze the polymorphism in the human apolipoprotein E gene. A method is described, in which the DNA is amplified by the polymerase chain reaction, immobilized, and sequenced by a semi-automatic procedure adaptable to clinical diagnosis. The three alleles of the apolipoprotein E gene, which differ from each other by two nucleotide substitutions and which influence serum cholesterol levels, were analyzed. The solid-phase method was able to resolve the correct nucleotide sequence in samples from both homozygous and heterozygous individuals. No cloning steps are needed and the immobilization and separation of the DNA is accomplished using magnetic beads.

Apolipoproteins E↗

PubMed1991

Colorimetric quantification of in vitro-amplified template DNA to be used for solid phase sequencing.

A protocol for colorimetric determination of DNA amplified by the polymerase chain reaction (PCR) and subsequently immobilized to a solid support is described. The protocol consists of three steps: (i) binding of PCR amplified lac operator-containing DNA to magnetic beads; (ii) binding of a Lac repressor-beta-galactosidase fusion protein to the lac operator and (iii) colorimetric detection of the immobilized beta-galactosidase. In practice, steps (i) and (ii) are performed concurrently. The protocol is well suited both for manual and automated procedures and the immobilized template can, after melting, be used directly for solid phase sequencing. The assay is used to demonstrate that template concentration is important for the quality of sequence data obtained from an automated DNA sequencer.

Base Sequence↗

PubMed1991

Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA.

A solid-phase approach is described for manual and automated sequencing of plasmid DNA obtained directly from bacterial colonies through the polymerase chain reaction. The DNA fragment is selectively immobilized to magnetic beads and after strand-specific elution, the eluted strand, as well as the remaining immobilized strand, is used for bidirectional dideoxy sequencing. The solid-phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The approach is exemplified by fluorescent sequencing of a cloned Streptomyces curacoi gene having a G + C content of more than 70%.

Base Sequence↗

PubMed1990

Solid phase in vitro mutagenesis using plasmid DNA template.

Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions. More than 80% mutants were obtained in both a single and a double primer approach. No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coli host cell. The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a site-specific restriction followed by a fill-in reaction. This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols.

Base Sequence↗

PubMed1990

Rapid detection and sequencing of specific in vitro amplified DNA sequences using solid phase methods.

We describe a rapid solid phase assay for detection and sequencing of DNA sequences based on selective introduction of biotin and isotope into the specific DNA fragment amplified by the polymerase chain reaction (PCR). A two-step PCR procedure is used to lower the background signal. The in vitro amplified material is immobilized on magnetic beads with covalently coupled streptavidin and the amount of bound label is measured. Samples identified as positive can be analysed by direct solid phase DNA sequencing. A strategy is also described to use general primers for detection, capturing and sequencing, which are not homologous to the specific sequence to be detected. The concept has been optimized using oligonucleotides specific for Staphylococci and Streptococci, respectively. Here, we show that the assay can be used for detection of Plasmodium falciparum in clinical samples.

Animals↗

PubMed1990

General colorimetric method for DNA diagnostics allowing direct solid-phase genomic sequencing of the positive samples.

A system for rapid colorimetric detection of specific genome DNA fragments amplified by the polymerase chain reaction (PCR) is described that has been designed to allow direct solid-phase sequencing of positive samples. The amplified material is immobilized on magnetic beads by using the biotin streptavidin system. An Escherichia coli lac operator DNA sequence is incorporated in the amplified material during the second step of a nested primer procedure. This 21-base-pair sequence is used for a general colorimetric detection with a fusion protein consisting of the E. coli Lac repressor and beta-galactosidase. Positive samples can be treated subsequently with alkali to obtain a single-stranded DNA template suitable for direct genomic sequencing. This method to detect immobilized amplified nucleic acids (DIANA) is well adapted for automated or semiautomated clinical assays. Here, we show that it can be used to detect and sequence Chlamydia trachomatis genomic DNA in clinical samples.

Amino Acid Sequence↗

PubMed1990

Direct cloning of the human genomic apolipoprotein E gene using magnetic separation of single-stranded DNA.

Solid-phase methods can be used for direct cloning of in vitro-amplified genomic DNA with high efficiency, without the need for restriction enzymes or ligase. Single-stranded DNA fragments are generated by magnetic separation and are simply mixed with single-stranded vector to form gap-duplex molecules that can be readily transformed. This strategy, in combination with direct solid-phase DNA sequencing, was used to analyze individual alleles in the human apolipoprotein E locus.

Amino Acid Sequence↗

PubMed1989

Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.

Base Sequence↗

PubMedDate not supplied

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.

Automation↗