Use of a PNA probe to block DNA-mediated PCR product formation in prokaryotic RT-PCR.
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.
Bender
Mikkel
M
Geological Survey of Denmark and Greenland, Copenhagen.
Holben
William E
WE
Sørensen
Søren J
SJ
Jacobsen
Carsten S
CS
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
D015342:DNA Probes; D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes; D020135:Peptide Nucleic Acids; D004247:DNA