Self-reporting PNA/DNA primers for PCR analysis

Genome Res. 2001 Apr;11(4):609-13. doi: 10.1101/gr.170401.

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.

MeSH terms

  • Chlamydia trachomatis / genetics
  • DNA Primers / genetics*
  • DNA, Bacterial / analysis
  • Endpoint Determination / methods
  • Fluorescent Dyes / analysis
  • Gene Amplification
  • Genes, Bacterial / genetics
  • Genes, ras / genetics
  • Humans
  • Neisseria gonorrhoeae / genetics
  • Peptide Nucleic Acids / genetics*
  • Polymerase Chain Reaction / methods*
  • Spectrometry, Fluorescence / methods

Substances

  • DNA Primers
  • DNA, Bacterial
  • Fluorescent Dyes
  • Peptide Nucleic Acids