A microtiter plate assay using cascade amplification for detection of nonisotopically labeled DNA.
We describe a microtiter-plate-based, colorimetric assay for DNA, the enzyme-linked DNA-enzyme-linked coagulation assay (EDNA-ELCA). The EDNA-ELCA uses amplification of the common pathway of coagulation for the ultrasensitive detection of DNA which is tagged by incorporation of functional groups such as biotin and fluorescein. The EDNA-ELCA enables detection of attomole amounts of DNA (< 1 pg per microtiter well), with a sensitivity 200-1000 times higher than other colorimetric techniques. The assay has been applied as an adjunct to PCR for quantitative determination of methicillin-resistant Staphylococcus aureus DNA at levels corresponding to 1-10(5) organisms. The EDNA-ELCA can also be used to assay DNA by hybridization; < 50 amol of an unlabeled DNA template is detected by hybridization to biotin- and fluorescein-labeled probes.
Rothschild
C B
CB
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157, USA.
Triscott
M X
MX
Bowden
D W
DW
Doellgast
G
G
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D003001:Cloning, Molecular; D003124:Colorimetry / Q000379:methods; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D017931:DNA Primers / Q000138:chemical synthesis*; D005340:Fibrinogen; D016650:Fluorescein-5-isothiocyanate; D006041:Goats / Q000276:immunology; D007202:Indicators and Reagents; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D000906:Antibodies; D017931:DNA Primers; D007202:Indicators and Reagents; D005340:Fibrinogen; D004247:DNA; D016650:Fluorescein-5-isothiocyanate