Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9743778 | Analytica Chimica Acta | 2005 | 7 Pages |
Abstract
Phosphorescent platinum(II)-coproporphyrin label (PtCP) was evaluated in post-PCR detection of nucleic acids by time-resolved fluorescence (TR-F) using three common formats. PtCP-labelled oligonucleotide primers and PtCP-dUTP were incorporated in a PCR to produce labelled amplified target â173 or 305Â bp DNA. Alternatively, aminoallyl-dUTP was incorporated in a PCR and the product was subsequently labelled with PtCP. The resulting PCR mixtures containing labelled dsDNA were separated on 1.5% agarose gels and then analysed by ethidium bromide staining and by direct detection of PtCP label on a commercial TR-F plate reader Victor2 (Perkin Elmer Life Sciences) used in scanning mode. In all cases label incorporation and high yields of amplified DNA were observed. Direct TR-F detection of PtCP-labelled DNA from a gel provided high sensitivity and signal to noise ratio, with limits of detection in the range of 9-22Â pg for all three formats. The sensitivity achieved with PtCP label was considerably better than that achieved with ethidium bromide staining (â¼1Â ng of dsDNA) or with conventional fluorescent label FITC. Neither the FITC label nor ethidium bromide staining interfered with PtCP detection, thus allowing multiplexed detection.
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Authors
Desmond J. O'Shea, Paul J. O'Sullivan, Gelii V. Ponomarev, Dmitri B. Papkovsky,