Evaluation of electrochemiluminescence- and bioluminescence-based assays for quantitating specific DNA.
The clinical value of PCR technology would be increased by development of improved quantitative methodology. Two new methods, electrochemiluminescence (ECL) and bioluminescence (BL), were evaluated for analytical dynamic range, sensitivity, and reproducibility of quantitation of specific DNA. The two assays were compared using an IL-2 template DNA amplified using one biotinylated forward primer and detected with sequence identical probes labeled in two different ways. PCR products were, captured on streptavidin-coated plates for BL and by streptavidin-coated beads for ECL. Product detection was accomplished using either a ruthenium (ECL) or a digoxigenin-labeled probe (BL). The ECL measurement was performed using the Perkin Elmer QPCR System 5000, while the BL methodology used a SeaLife Science AquaLite Aequorin-antibody conjugate, which was detected with a ML3000 luminometer. Both instruments were found to be extremely sensitive with accurate quantitation of label in the attomole range, allowing detection during the exponential phase of PCR amplification. In our hands, it was possible to detect 1.5 x 10(14) copies (18 cycles) of IL-2 PCR product using ECL and 1 x 10(13) copies (14 cycles) using BL technology. Overall, we found the BL assay to be a rapid, sensitive, and inexpensive way to quantitate PCR-generated products with a broad range of potential analytical applications.
Siddiqi
A M
AM
Department of Pathology, Emory University, Atlanta, Georgia 30022, USA.
Jennings
V M
VM
Kidd
M R
MR
Actor
J K
JK
Hunter
R L
RL
D000331:Aequorin / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D015342:DNA Probes; D004274:DNA, Recombinant / Q000032:analysis / Q000235:genetics; D004076:Digoxigenin / Q000031:analogs & derivatives / Q000378:metabolism; D007376:Interleukin-2 / Q000235:genetics; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction; D012428:Ruthenium / Q000378:metabolism; D019809:Streptavidin
D001426:Bacterial Proteins; D017931:DNA Primers; D015342:DNA Probes; D004274:DNA, Recombinant; D007376:Interleukin-2; D000331:Aequorin; D001710:Biotin; D012428:Ruthenium; D004247:DNA; D019809:Streptavidin; D004076:Digoxigenin