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A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

✍ Scribed by Yu, Hao ;Bruno, John G. ;Cheng, Tu-chen ;Calomiris, Jon J. ;Goode, Michael T. ;Gatto-Menking, Deborah L.

Publisher
John Wiley and Sons
Year
1995
Weight
594 KB
Volume
10
Category
Article
ISSN
0884-3996

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✦ Synopsis

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture o f biotinylated and ruthenium(l1) trisbipyridal (R~(bpy),~+)-end-labelled primers. In this way, biotin for capture and Ru(bpy)32+ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR o f a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail o f biotin-and Ru(bpy):,*+-labelled primers amplified a 1 kilobase region. Serial dilution o f PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay o f probe hybridization t o a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection o f DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 t o 100 times more sensitive than conventional ethidium bromide staining. The combination o f DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method o f quantitating DNA which, owing t o its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.

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