We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.

Nucleohistone solubilized from rabbit thymus nuclei by an endogenous nuclease has in 0.15 M salt an exceptionally low intrinsic viscosity and very high sedimentation velocity. A fully reversible expansion of configuration occurs on lowering ionic strength. When [eta] is plotted against I-1/2 and extrapolated to high I, [eta] = 0 is reached at I = 0.4-1 M and [eta] at I = infinity is negative, contrary to the behavior of DNA and of the great majority of polyelectrolytes, which extrapolate to a positive [eta] at I = infinity. This behavior demands that the configuration of nucleohistone depends not only on electrostatic expansive forces but also on contracting forces which are not electrostatic and do not go to zero in any accessible configuration. Intramolecular hydrophobic bonds might provide such contracting forces. Increasing I above 0.15 M leads to precipitation near 0.3 M and redissolution with dissociation of F1 and expansion in 0.6 M. The expansion is largely but not completely reversed on return to 0.15 M. Much further expansion occurs in I = 1.2 M. Nucleohistone exposed to 1.2 M could not be redissolved in the original medium. Nucleohistone depleted of F1 exhibits a similar expansion as ionic strength is reduced, at higher viscosities throughout. On extrapolation to I = infinity both positive and negative viscosities were observed, on different lots, perhaps reflecting variable extraction of other histones. Circular dichroism spectra are very little affected by ionic strength (0.6 M and lower) or F1 removal, despite tenfold changes in viscosity.

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