Affinity capture and recovery of DNA at femtomolar concentrations with peptide nucleic acid probes.
The efficacy of PNA vs DNA oligomers for the recovery of femtomolar concentrations of 16S rDNA targets was determined with solution- and mixed-phase hybridization formats and limiting dilution quantitative PCR. Several results contradict existing perceptions of expected PNA behavior deduced from hybridization studies with oligonucleotide targets at high concentration. For example, DNA probes in the solution hybridization format performed as well as or better than PNA probes under high- or low-salt conditions, regardless of hybridization time or target size. In the mixed-phase hybridization format, however, PNA probes showed certain advantages, with more rapid and efficient binding/recovery of target nucleic acids regardless of target size. Recovery of target DNA with PNA probes was always more efficient in low-salt (20 mM in Na(+)) than high-salt (400 mM in Na(+-)) phosphate buffer. Recovery of target DNA by PNA probes was enhanced in the presence of excess, nontarget DNA, and differences in PNA efficacy under low- or high-salt conditions vanquished. In contrast, DNA probe performance was unaffected by the presence or absence of exogenous DNA in both solution- and mixed-phase hybridization formats. The absolute recovery and detection limit of the affinity purification method with either DNA or PNA probes was approximately 10(2) input target molecules at zeptamolar concentrations.
Chandler
D P
DP
Environmental Microbiology Group, Statistical Resources Group, Pacific Northwest National Laboratory, 900 Battelle Boulevard, Mail Stop P7-50, Richland, Washington 99352, USA. dp.chandler@pnl.gov
Stults
J R
JR
Anderson
K K
KK
Cebula
S
S
Schuck
B L
BL
Brockman
F J
FJ
D015342:DNA Probes; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics
D015342:DNA Probes; D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D020135:Peptide Nucleic Acids; D012336:RNA, Ribosomal, 16S