| 摘要 |
An improved method is provided for making a dimer of a first nucleic acid, terminally derivatized with a moiety of formula -(OPO2)NH(R11)SS(R12)NH2, and a second nucleic acid, terminally derivatized with a moiety of formula -(OPO2)NH(R21)SS(R22)NH2, wherein R11, R12, R21 and R22 are the same or different and each is an alkylene group of 2 to 20 carbon atoms or a cycloalkylene group of 3 to 20 carbon atoms. The improvement entails reducing the disulfide bonds of the two nucleic acids and, prior to oxidation of the resulting thiol adducts to form dimers, dialyzing from the nucleic acid solution the compounds of formulae HS(R12)NH2 and HS(R22)NH2, made upon reduction of the disulfides, while maintaining the concentration of reducing agent in the solution sufficiently high to maintain the concentrations of the nucleic acid thiol adducts. In a preferred embodiment, after removal of most of the compounds of formulae HS(R12)NH2 and HS(R22)NH2 from the nucleic acid solution, reducing agent is removed from said solution by dialysis and the nucleic acid thiol adducts are dimerized by oxidation with atmospheric oxygen. The nucleic acids are preferably derivatized at the 5'-carbons of their 5'-termini. The preferred groups R11, R12, R21 and R22 are -(CH2)2-. The preferred reducing agent is dithiothreitol. In a preferred application of the invention, one of the derivatized nucleic acids is a replicative RNA and the other is an oligonucleotide with the sequence of an affinity molecule for a nucleic acid analyte to be detected in a nucleic acid probe hybridization assay. |
