Biomedical subjects

J Lundeberg

Publications and source records attributed to J Lundeberg.

97 records · Page 6Linked to original sources

PubMed1991

Analysis of the V3 loop in neutralization-resistant human immunodeficiency virus type 1 variants by direct solid-phase DNA sequencing.

To study the molecular basis for the emergence of human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to neutralization by autologous sera, the DNA sequence of the envelope V3 loop was determined in HIV-1 isolates derived from four patients with primary HIV-1 infection and sequentially thereafter. The gene fragment encoding the V3 loop of gp120 was amplified by a nested polymerase chain reaction (PCR) and subsequently sequenced by a novel solid phase DNA sequencing approach allowing direct sequencing of the viral genome without the need for previous cloning. The results show that all patients have HIV-1 with unique primary sequence of the V3 loop and antibodies to this structure are produced at seroconversion. The structural analysis also demonstrates that the mechanism for virus escape from neutralization in vivo is complex. Thus, in one patient the emergence of neutralization-resistant viruses coincided with the introduction of several amino acid changes in the V3 loop, while in two other patients the V3 loop remained completely unchanged. These findings suggest that an understanding of the interaction between the humoral immune response and HIV-1 may require detailed structural studies of the entire envelope.

Amino Acid Sequence↗

PubMed1991

Rapid colorimetric quantification of PCR-amplified DNA.

A diagnostic system for rapid colorimetric quantification of the initial amount of DNA template amplified by the polymerase chain reaction is described. The method is based on co-amplification of target DNA with a cloned DNA fragment, in which a lac operator sequence has been introduced by in vitro mutagenesis. The in vitro-amplified material is immobilized on magnetic beads using the biotin-streptavidin system, and the ratio of target DNA to cloned mutated DNA can be determined using a fusion protein consisting of the Escherichia coli LacI repressor and beta-galactosidase. This method for quantitative detection of immobilized amplified nucleic acids is well adapted for rapid automated or semi-automated assays. Here, we show that it can be used to detect and quantify Plasmodium falciparum genomic DNA in clinical samples.

Animals↗

PubMed1990

Affinity purification of specific DNA fragments using a lac repressor fusion protein.

A new method for purification of specific DNA sequences using a solid phase technique has been developed based on a fusion between the Escherichia coli lac repressor gene (lacI) and the staphylococcal protein A gene (spa). The fusion protein, expressed in Escherichia coli, is active both in vivo and in vitro with respect to its three functional activities (DNA binding, IPTG induction, and IgG binding). The recombinant protein can be immobilized in a one-step procedure with high yield and purity using the specific interaction between protein A and the Fc-part of immunoglobulin G. The immobilized repressor can thereafter be used for affinity purification of specific DNA fragments containing the lac operator (lacO) sequence.

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

Rapid colorimetric detection of in vitro amplified DNA sequences.

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.

Animals↗

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↗