Biomedical subjects

M S Ellwood

Publications and source records attributed to M S Ellwood.

3 recordsLinked to original sources

PubMed1988

A novel diagnostic method based on DNA strand displacement.

A novel approach for the detection of specific DNA or RNA sequences based on branch migration and DNA strand displacement is described. A partially double-stranded probe complex is prepared with a detectable label on one of the two strands and incubated with analyte molecules under hybridization conditions. The analyte molecules hybridize to the single-stranded portion of the probe complex and undergo branch migration to release the labelled DNA strand from the complex. Initial characterization of the assay indicates that both qualitative and quantitative information about analytes present in a sample can be obtained. The strand displacement assay is more sensitive to sequence alterations in the analyte than is a hybridization assay and can be promoted by rec A protein at 37 degrees C. Finally, a method for preparing probe complexes by cloning in a single-stranded DNA vector is also described.

DNA↗

PubMed1986

Strand displacement applied to assays with nucleic acid probes.

This novel method for the detection of specific nucleic acid sequences has potential applications to clinical diagnosis. During hybridization, a signal-bearing nucleic acid strand is displaced by the target nucleic acid from a partially single-stranded complementary probe strand of nucleic acid. The probe:signal strand complex is prepared by hybridizing single-stranded probe that is entirely complementary to the target nucleic acid with a shorter signal sequence that is complementary to a portion of the probe strand. The sample nucleic acid is added to this hybrid complex under hybridization conditions. The target sequence, if present in the sample, will hybridize first to the unoccupied probe sequences, and then will displace the labeled strand by branch migration. By this "strand displacement" the signal strands are freed in solution, where they may be separated from those still hybridized; the quantity of label measured is directly proportional to the amount of analyte sequences in the sample. This method, demonstrated here for model and synthetic DNAs, can easily be adapted for the detection of any RNA or DNA sequence and obviates the need for immobilization of sample. A wide variety of labeling techniques can be used, and the displacement can be performed in solution or with the hybrid complex attached to a solid support. This assay circumvents nonspecific binding of label to the filter matrix and the laborious washing steps inherent in other assays involving nucleic acid probes.

Animals↗

PubMed1984

Differential regulation of the 70K heat shock gene and related genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains a family of genes related to Hsp70, the major heat shock gene of Drosophila melanogaster. The transcription of three of these genes, which show no conservation of sequences 5' to the protein-coding region, was analyzed. The 5' flanking regions from the three genes were fused to the Escherichia coli beta-galactosidase structural gene and introduced into yeasts on multicopy plasmids, putting the beta-galactosidase production under yeast promoter control. Analysis of beta-galactosidase mRNA and protein production in these transformed strains revealed that transcription from the three promoters is differentially regulated. The number of transcripts from one promoter is vastly increased for a brief period after heat shock, whereas mRNA from another declines. Transcripts from a third gene are slightly enhanced upon heat shock; however, multiple 5' ends of the mRNA are found, and a minor species increases in amount after heat shock. Transcription of these promoters in their native state on the chromosome appears to be modulated in the same manner.

Base Sequence↗