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. Many results contradict existing perceptions of expected PNA behavior deduced from hybridization studies with oligonucleotides and high target concentrations. 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, non-target 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 102 input target molecules at zeptamolar concentrations.
Revised: October 9, 2000 |
Published: October 1, 2000
Chandler D.P., J.R. Stults, K.K. Anderson, S.T. Cebula, B.L. Schuck, and F.J. Brockman. 2000."Affinity capture and recovery of DNA at femtomolar concentrations with peptide nucleic acid probes."Analytical Biochemistry 283, no. 2:241-249.PNNL-SA-32768.