A comparative study of PCR product detection and quantitation by electro-chemiluminescence and fluorescence.
Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)3(2+))-end-labelled primers. In this way, biotin for capture and Ru(bpy)3(2+) for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)3(2+)-labelled primers amplified a 1 kilobase region. Serial dilution of 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 of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.
Optech Corporation, San Antonio, TX 78229, USA.
H
H
Yu
J G
JG
Bruno
T C
TC
Cheng
J J
JJ
Calomiris
M T
MT
Goode
D L
DL
Gatto-Menking
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004996:Ethidium; D005453:Fluorescence; D005456:Fluorescent Dyes; D015750:Genes, gag; D006088:Gram-Negative Aerobic Bacteria / Q000235:genetics; D006678:HIV / Q000235:genetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004279:DNA, Viral; D005456:Fluorescent Dyes; D004996:Ethidium