The application of nucleic acid chemistry to studies on the functional organization of gene control regions.
A new, extremely high yield method for synthesizing deoxyoligonucleotides has been developed. Syntheses are completed on a silica gel polymer support using appropriately protected deoxymononucleotide 3'-phosphoramidites. These mononucleotide intermediates are synthesized using simple organo chemical procedures and can be stored indefinitely as dry powders. Activation is achieved by adding a weak acid such as tetrazole to an acetonitrile solution of the mononucleotide. Chemical steps for the addition of one nucleotide to the support are the following: (1) detritylation using ZnBr2 in nitromethane/methanol (5 min); (2) condensation of a 5'-O-dimethoxytrityldeoxynucleoside 3'-methoxy-N, N-dimethylamino phosphine with the support bound nucleoside (5 min); (3) blocking unreacted, support bound nucleoside hydroxyl groups with acetic anhydride (5 min); (4) oxidation of the phosphite to the phosphate with I2 (2 min). Completed deoxyoligonucleotides are isolated by first sequentially treating with thiophenol, t-butylamine, ammonium hydroxide, and 80% acetic acid and then purifying to homogeneity by electrophoresis on polyacrylamide gels. The method is extremely fast (approximately 30 min per cycle), yields in excess of 98% per condensation are obtained, and isolation of the final product is a simple, one step purification. This methodology has been used to synthesize several lac operators containing altered sequences. The results of binding studies indicate that lac repressor does not recognize a unique DNA sequence.
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D005796:Genes; D007202:Indicators and Reagents; D008722:Methods; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D009841:Oligonucleotides / Q000138:chemical synthesis*; D058428:Silica Gel; D012822:Silicon Dioxide
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D058428:Silica Gel; D012822:Silicon Dioxide; D004247:DNA