Use of a PNA probe to block DNA-mediated PCR product formation in prokaryotic RT-PCR

Biotechniques. 2007 May;42(5):609-10, 612-4. doi: 10.2144/000112437.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Infections / diagnosis
  • DNA / genetics*
  • DNA Probes / metabolism*
  • DNA, Bacterial / analysis
  • DNA, Bacterial / genetics
  • Escherichia coli / genetics
  • Escherichia coli / isolation & purification
  • Genes, Bacterial
  • Nucleic Acid Heteroduplexes
  • Peptide Nucleic Acids / metabolism*
  • Prokaryotic Cells / metabolism*
  • Pseudomonas putida / genetics
  • Pseudomonas putida / isolation & purification
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Salmonella typhimurium / genetics
  • Salmonella typhimurium / isolation & purification

Substances

  • DNA Probes
  • DNA, Bacterial
  • Nucleic Acid Heteroduplexes
  • Peptide Nucleic Acids
  • DNA