PubMed HealthSearch

PubMed · 1993334

Simplified format for DNA probe-based tests.

Abstract

The approach I describe to using DNA probes in diagnostic tests is simpler than most existing formats. DNA in a sample is labeled by chemical reaction with bisulfite and methylamine to generate a sulfonated derivative. The DNA need not be purified to do this. The labeled sample is then incubated with an unlabeled, purified probe DNA, which is immobilized to a solid support. The amount of label remaining on the solid support after washing is detected by a monoclonal antibody that recognizes modified cytosines. The intensity of the signal depends on the amount of target DNA in the sample. Detection limits depend on the amount of immobilized DNA and on the degree of physical entrapment of the labeled DNA in sample material, but can be as low as 5 pg. This format is well suited to automation for use with existing robotic enzyme immunoassay procedures.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

W Bains. 1991. Simplified format for DNA probe-based tests.. https://pubmed.ncbi.nlm.nih.gov/1993334/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

High-throughput method for detecting genomic-deletion polymorphisms.

DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.

DNA Probes

Female twin with Hunter disease due to nonrandom inactivation of the X-chromosome: a consequence of twinning.

We report the occurrence of Hunter disease (mucopolysaccharidosis type II) in a karyotypically normal girl who was one of identical twins. Molecular studies showed nonrandom X-inactivation in both her fibroblasts and lymphocytes, while her normal twin showed equal usage of both X chromosomes. In view of previous reports of 7 pairs of identical female twins in which one had Duchenne muscular dystrophy, it seems that twinning may be strongly associated with nonrandom X-inactivation, and is not specific to the properties of the disease causing gene.

DNA Probes