The phosphorous amide approach is of the highest coupling efficiency among the DNA in situ synthesis approaches at present. The strict anhydrous condition is required in the process of DNA in situ synthesis, and the DNA synthesis in the molecular stamp method must be performed in the anhydrous glove box. Because various volatile reagents remain in the glove box, the reagent gases invade into the coupling reaction system so that the coupling efficiency is decreased. This decrease is caused by the pyridine contained in the oxidizing agent. An improved oxidizing agent system of I 2/Ac 2O/AcOH/THF for the synthesis of oligodeoxy-nucleotides was reported. Using this oxidizing agent, 16-mer oligodeoxynucleotide probes were successfully synthesized on the CPG(controlled pore size glass) or the modified glass slide and a single step coupling efficiency of 98.2% was achieved. The synthesized DNA probes were hybridized with fluorescence and gold-labeled target oligodeoxynucleotides respectively. The fluorescence intensity and gray level for the synthesized probes in the I 2/Ac 2O/AcOH/THF oxidizing agent were similar to those in the traditional phosphorous amide approach possessing a water- and pyridine-containing oxidizing agent I 2/H 2O/Pyridine/THF. The reported oxidizing agent will make the contact printing approach to in situ synthesized DNA microarrays more feasible by eliminating the decrease of coupling efficiency which is because the cross reagent interfering arises from pyridine and water.
Microporous polyamide-6 membranes were hydrolyzed in 0.01 mol/L NaOH/CH 3OH medium by refluxing for about 36 h. By investigating the UV spectra, the hydrolyzing conditions were optimized. A pH value of 12 and reaction time of 36 h are the preferred conditions for hydrolysis. The treated membrane can be applied for in site synthesis of oligonucleotide and, for example, the oligonucleotide probes of 5′-AACCACCAAACACAC-3′ were successfully synthesized on the hydrolyzed membrane,which was proved by the high resolution X-ray photoelectron spectroscopy(XPS) of P 2p. The single step coupling efficiency was determined by UV spectra to be in excess of 98 %.
