DNA enzyme immunoassay: a rapid and convenient colorimetric method for diagnosis of cystic fibrosis.
Genetic analysis of inherited diseases has been greatly facilitated by new approaches, involving genomic DNA amplification by the polymerase chain reaction (PCR), followed by hybridization with wild type-specific or mutation-specific oligonucleotide (MSO) probes. The main advantage of these methods is that they allow easy detection of point mutations starting from minimal amounts of biological materials. These techniques, however, require procedures which are not well suited to large-scale screening or use in routine laboratories. The development of dedicated kits to perform these tests efficiently in clinical laboratories is an important current issue. We developed a new non-radioisotopic assay to reveal specifically DNA-DNA hybrids between amplified DNA and MSO probes, and applied it to the detection of two mutations causing cystic fibrosis. The detection of hybrids is achieved by means of an anti double-stranded DNA antibody, in a format which is designed as a colorimetric assay resembling a common enzymatic immunoassay (EIA). The assay detects the hybridization event, independent of the nucleic acid sequences involved in the formation of the specific hybrids, and can be used with any combination of target DNA and probes. Therefore, this test represents a significant improvement for the clinical use of the polymerase chain reaction in the diagnosis of inherited diseases.
Mazza
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Consiglio Nazionale delle Ricerche (CNR), Medical School, University of Brescia, Italy.
Mantero
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G
Primi
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D
D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001710:Biotin; D003124:Colorimetry / Q000379:methods*; D003550:Cystic Fibrosis / Q000175:diagnosis*; D004247:DNA / Q000097:blood* / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D015345:Oligonucleotide Probes; D001710:Biotin; D004247:DNA