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.
Footnotes
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↵1 Corresponding author.
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E-MAIL jcoull{at}bostonprobes.com; FAX 781-276-4931.
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Article and publication are at www.genome.org/cgi/doi/10.1101/gr.170401.
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- Received November 11, 2000.
- Accepted February 8, 2001.
- Cold Spring Harbor Laboratory Press
