PNNL

Abstract

Bis-PNAs (PNA clamps), PNA oligomers and DNA oligonucleotides were evaluated as affinity purification reagents for sub-femtomolar 16S rDNA and rRNA targets in soil, sediment and industrial air filter nucleic acid extracts. Under a high salt buffer condition (200 mM NaPO4, 100 mM Na2EDTA, 2% SDS), the DNA probe recovered as much or more target DNA than both types of PNA probes. PNA probe efficacy was significantly enhanced in the presence of excess, non-target DNA, and in a 'low salt' extraction/hybridization buffer. Recovery and detection efficiencies for target DNA concentrations > 100 pg were generally > 20%, but depended upon the specific probe, solution background and salt condition. The DNA probe had a lower absolute detection limit of 100 fg target (830 zM; zeptamole = 10-21 mole) in high salt buffer. In the absence of exogenous DNA (e.g. soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit, even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification/detection (830 zM) than the DNA probe. In varied environmental samples, the rank order for capture probe performance in high salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA probe generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of non-target DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture probe depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA) and detection objective.