Self-reporting PNA/DNA primers for PCR analysis

Abstract

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.