Non-radioisotopic methods for DNA probes.
We have recently developed a new colorimetric method, DNA enzyme immunoassay (DEIA), for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single-stranded DNA. The molecule does not react with a specific probe immobilized on microwells through an avidin-biotin bridge, nor with non-specific amplified sequences, since they are removed by washing. The antibody reveals the hybridization event, independently of the DNA sequences and, for this reason, the method is broadly applicable and extremely versatile. Although we chose a format based on the immobilization of the probe through an avidin-biotin interaction, DEIA assay can also be applied to other analytical schemes that do not require any modification of the probe. Most importantly, the test has an ELISA format and is rapid and convenient for processing large numbers of samples. This technology has been applied, in our laboratory, to different areas, including virology, genetic and basic immunology. The DEIA assay has been successfully used to detect the presence of hepatitis B (HBV), hepatitis C (HCV) and hepatitis delta virus (HDV) sequences in serum of patients, to discriminate different HLA alleles, to identify mutations in the Cystic Fibrosis gene, and to investigate the role of the T cell receptor in some immunological diseases. The results obtained in all our experiments demonstrated that the advantages offered by the assay do not penalize its analytical performance as compared to conventional Southern blot.
Imberti
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L
Consorzio per le Biotecnologie, Consiglio Nazionale delle Ricerche (CNR), Laboratorio di Biotecnologie, Brescia, Italy.
Sottini
A
A
Bettinardi
A
A
Mazza
C
C
Albertini
A
A
Primi
D
D
D003124:Colorimetry / Q000379:methods; D003550:Cystic Fibrosis / Q000235:genetics; D015342:DNA Probes / Q000032:analysis*; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D007154:Immune System Diseases / Q000276:immunology; D007124:Immunoenzyme Techniques; D011948:Receptors, Antigen, T-Cell / Q000276:immunology
D015342:DNA Probes; D011948:Receptors, Antigen, T-Cell