ABSTRACT
A low voltage (180-V) dc glow discharge device was inserted just below the pneumatic nebulizer in a particle beam interface of a high performance liquid chromatography/mass spectrometry system. The discharge in a helium atmosphere increased the signal produced by 12 test compounds by factors of about 2-6. The increases in signal were probably produced by an increase in the efficiency of solute transmission through the interface. The signal increases caused by the glow discharge were compared to somewhat larger increases caused by 0.01-M ammonium acetate in the mobile phase. The combination of glow discharge and ammonium acetate provided no meaningful advantage over the individual techniques. The mechanism of improved transport efficiency is not proven, but the neutralization of charged particles is a viable hypothesis.
ABSTRACT
Sequence-specific spin-labeled oligodeoxynucleotides with conformation-sensitive electron paramagnetic resonance (EPR) signals are synthesized and examined as solution-phase nucleic acid hybridization probes. Either a proxyl or tempo ring linked to the C(5) position of deoxyuridine (dU) by a nonrigid two-atom methylamino tether is incorporated within 15-mers by phosphotriester chemistry yielding stable spin-labeled probes with distinctive EPR specific activity (AEPR) values. The AEPR is greater for a proxyl-labeled than for a tempo-labeled probe and is consistent with EPR data of enzymatically labeled 26-mers [Bobst, A. M., Pauly, G. T., Keyes, R. S., and Bobst, E. V. (1988) FEBS Lett. 228, 33-36], after normalizing for percent labeling. The spectral characteristics of the free probes and the probe/target complexes are similar to those of enzymatically spin-labeled nucleic acids containing a different nonrigid two-atom-tethered spin label [Bobst, A. M., Kao, S.-C., Toppin, R. C., Ireland, J. C., and Thomas, I. E. (1984) J. Mol. Biol. 173, 63-70]. The presence of target DNA is detected in solution by EPR spectroscopy and the assay is based on the characteristic line-shape change associated with hybridization. The EPR spectra of free and bound probe reflect little interference from changes in global dynamics of the probe, and the line-shape change upon complexation results primarily from a change in local base dynamics. The presence or absence of hybridization can be detected in a loop-gap resonator with about 1 pmol of spin-labeled 15-mer within minutes.