Self-reporting PNA/DNA primers for PCR analysis.
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.
Fiandaca
M J
MJ
Boston Probes, Bedford, Massachusetts 01730, USA.
Hyldig-Nielsen
J J
JJ
Gildea
B D
BD
Coull
J M
JM
D002692:Chlamydia trachomatis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D023381:Endpoint Determination / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis; D005784:Gene Amplification; D005798:Genes, Bacterial / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D009344:Neisseria gonorrhoeae / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods
D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids