Direct analysis of polymerase chain reaction products using enzyme-linked immunosorbent assay techniques

• Published in: Analytical biochemistry Vol. 198; no. 1; pp. 86 - 91
• Main Authors: Landgraf, Axel, Reckmann, Bernd, Pingoud, Alfred
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.10.1991; Elsevier
• Subjects: Avidin; Bacterial Proteins; Base Sequence; Biological and medical sciences; Biotin; DNA, Single-Stranded; Enzyme-Linked Immunosorbent Assay - methods; Fluorescein-5-isothiocyanate; Fundamental and applied biological sciences. Psychology; Horseradish Peroxidase; Molecular and cellular biology; Molecular genetics; Molecular Probe Techniques; Molecular Sequence Data; Polymerase Chain Reaction - methods; Proto-Oncogenes; Streptavidin; Characterization; Human; Polymerase chain reaction; ELISA assay; DNA; Blood
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(91)90510-Z

PCR products obtained using primers carrying at their 5′ ends biotin and an antigenic group (e.g., fluorescein) can be quantitatively analyzed by immunological techniques. The procedure described here does not require electrophoretic separation and/or hybridization with radioactive probes. It takes advantage of the fact that biotinylated DNA can be immobilized on avidin- or streptavidin-coated microtiter plates and then can be quantitated by an ELISA specific for the antigenic group. The PCR/ELISA procedure is suitable for routine diagnostic purposes and lends itself to automation. The sensitivity of the immunological detection system that employs horseradish peroxidase linked to antifluorescein antibodies is high: 1 μl of the PCR mixture obtained after approximately 25 cycles of amplification of 1 ng/μl genomic template DNA is sufficient for the detection of human single-copy genes. The usefulness of the procedure for the quantitative analysis of the amount of DNA present in a blood or tissue sample is discussed.