title: Use of a PNA probe to block DNA-mediated PCR product formation in prokaryotic RT-PCR.
abstract: A novel method eliminating DNA-mediated PCR product formation in reverse transcription PCR (RT-PCR) amplification of specific RNA sequences is described. The method exploits the higher melting temperature values of peptide nucleic acid (PNA)/DNA duplexes compared with DNA/DNA duplexes by binding a sequence-specific PNA probe to a genomic sequence immediately overlapping one of the PCR-primer attachment sites within the sequence of interest. Hybridization of the blocking probe precludes primer attachment to DNA without affecting attachment of the same primer to the reverse transcription-generated cDNA sequence. A four-step PCR cycle is used that allows the PNA probe to hybridize to the DNA strand at a higher temperature just prior to the primer annealing step.
authors.affiliation: Geological Survey of Denmark and Greenland, Copenhagen.
authors.forename: Mikkel
authors.initials: M
authors.lastname: Bender
authors.forename: William E
authors.initials: WE
authors.lastname: Holben
authors.forename: Søren J
authors.initials: SJ
authors.lastname: Sørensen
authors.forename: Carsten S
authors.initials: CS
authors.lastname: Jacobsen
mesh_terms: D001424:Bacterial Infections / Q000175:diagnosis; D004247:DNA / Q000235:genetics*; D015342:DNA Probes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D009692:Nucleic Acid Heteroduplexes; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D011387:Prokaryotic Cells / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics / Q000302:isolation & purification
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes; D020135:Peptide Nucleic Acids; D004247:DNA