Direct analysis of polymerase chain reaction products using enzyme-linked immunosorbent assay techniques.
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 anti-fluorescein antibodies is high: 1 microliter of the PCR mixture obtained after approximately 25 cycles of amplification of 1 ng/microliter 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.
Landgraf
A
A
Abteilung Biophysikalische Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, Germany.
Reckmann
B
B
Pingoud
A
A
D001360:Avidin; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D004277:DNA, Single-Stranded; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D006735:Horseradish Peroxidase; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011519:Proto-Oncogenes; D019809:Streptavidin
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D001360:Avidin; D001710:Biotin; D019809:Streptavidin; D006735:Horseradish Peroxidase; D016650:Fluorescein-5-isothiocyanate