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

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

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Geological Survey of Denmark and Greenland, Copenhagen.


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.

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