Quantification of polymerase chain reaction products: enzyme immunoassay based systems for digoxigenin- and biotin-labelled products that quantify either total or specific amplicons.
Enzyme immunoassays were developed for quantification of polymerase chain reaction (PCR) products, referred to as amplicons. Amplicons were dual labelled simultaneously by enzymatic incorporation of digoxigenin and biotin during PCR. For total amplicon quantification, Microfluor B polystyrene wells, compatible with chemiluminescent detection, were coated with streptavidin. Dual labelled amplicons were bound, treated with anti-digoxigenin antibody conjugated to alkaline phosphatase to complete the two-site sandwich immunoassay configuration, and detected by the chemiluminescence generated upon hydrolysis of a phosphate substituted dioxetane substrate, AMPPD. For specific amplicon quantification, the Microfluor B wells were coated with an unlabelled DNA probe complementary to the labelled amplicon target. Subsequent steps were performed as described above. This assay detects 2 pg of specifically amplified DNA. Chemiluminescent detection provides a linear range of four orders of magnitude for amplicon quantification. The non-radioactive labels are safe and stable. PCR as described here obviates the need for labelled primers and constitutes the initial report of concurrent dual non-radioactive labelling of DNA by a DNA polymerase.
Stevens
J
J
New England Medical Center, Pulmonary and Critical Care Division, Boston, MA 02111, USA.
Yu
F S
FS
Hassoun
P M
PM
Lanzillo
J J
JJ
D000818:Animals; D001483:Base Sequence; D001710:Biotin; D002417:Cattle; D004247:DNA / Q000032:analysis* / Q000235:genetics; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin; D004727:Endothelium; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D014969:Xanthine Oxidase / Q000235:genetics
D003856:Deoxyuracil Nucleotides; D001710:Biotin; D004247:DNA; D014969:Xanthine Oxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007703:Peptidyl-Dipeptidase A; D004076:Digoxigenin