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Vol. 11, Issue 4, 609-613, April 2001

METHODS
Self-Reporting PNA/DNA Primers for PCR Analysis

Mark J. Fiandaca, Jens J. Hyldig-Nielsen, Brian D. Gildea, and James M. Coull1

Boston Probes, Bedford, Massachusetts 01730, USA

We report a new fluorogenic method for sealed-tube PCR analysis using a quencher-labeled peptide nucleic acid (Q-PNA) probe. The Q-PNA hybridizes to a complementary tag sequence located at the 5' end of a 5' fluorophore-labeled oligonucleotide primer, quenching the primer's fluorescence. Incorporation of the primer into a doublestranded amplicon causes displacement of the Q-PNA such that the fluorescence of the sample is a direct indication of the amplicon concentration. The Q-PNA is able to quench multiple primers bearing distinct 5' fluorophores in a single reaction. We show realtime quantitative detection of a single-copy gene, K-ras, from human genomic DNA, as well as an endpoint multiplex assay for Chlamydia trachomatis and Neisseria gonorrhoeae targets. Because the Q-PNA may be used to quench any primer that contains the 5' tag sequence, it is possible to inexpensively adapt an existing primer set for use in a self-reporting fluorescent assay by including the tag sequence in one of the primers.

1 Corresponding author.

11:609-613 ©2001 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/01 $5.00
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