Open AccessEvaluation of electrochemiluminescence‐ and bioluminescence‐based assays for quantitating specific DNA
Author(s) -
Siddiqi Anwer M.,
Jennings Veronica M.,
Kidd Marybeth R.,
Actor Jeffrey K.,
Hunter Robert L.
Publication year - 1996
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/(sici)1098-2825(1996)10:6<423::aid-jcla19>3.0.co;2-z
Subject(s) - bioluminescence , electrochemiluminescence , luminescent measurements , chemistry , computational biology , biochemistry , microbiology and biotechnology , biology , chromatography , materials science , detection limit , optoelectronics , luminescence
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 SeaLite 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 1.5×10 14 copies (18 cycles) of IL‐2 PCR product using ECL and 1×10 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. © 1996 Wiley‐Liss, Inc.