Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps.
Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =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 of target (830 zM [1 zM = 10(-21) M]) 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 and detection (830 zM) than the DNA oligomer. 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 oligomer 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 nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.
Environmental Microbiology Group, Pacific Northwest National Laboratory, Richland, Washington 99352, USA. dp.chandler@pnl.gov
D P
DP
Chandler
J R
JR
Stults
S
S
Cebula
B L
BL
Schuck
D W
DW
Weaver
K K
KK
Anderson
M
M
Egholm
F J
FJ
Brockman
D002846:Chromatography, Affinity; D015342:DNA Probes; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020564:Deltaproteobacteria / Q000235:genetics* / Q000302:isolation & purification; D004783:Environmental Microbiology; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004275:DNA, Ribosomal; D020135:Peptide Nucleic Acids; D012336:RNA, Ribosomal, 16S