SUMMARYA method for the microscale (one m o l e ) synthesis of volatile, 14C-labelled acid chlorides from Ba14C03 using vacuum line techniques is described. By a judicious choice of reagents, desired intermediates and products may be isolated in high yield by simple vacuum transfer techniques without fractionation.
Background: Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics.
Methods: Luminescent oxygen channeling assay (LOCITM) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types.
Results: In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets.
Conclusions: This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.
We describe herein, a novel synthesis of the previously unreported compound, 4,6‐dibromo‐2‐pyridone (3) and its use in the preparation of [3H]‐RS‐91309, a potassium channel modulator. This key intermediate, (3), was reduced with carrier free tritium gas to furnish [4,6‐3H]‐2‐pyridone (9) having a specific activity of 50 Ci/mmole. Condensation of (9) with epoxide (10), followed by elaboration of the resulting chromene methyl group of (12) gave [3H]‐RS‐91309 (14) whose specific activity was also 50 Ci/mmole. This chemistry, as well as the solution of several microscale related stoichiometry problems is discussed.
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