Non-nucleosidic phosphoramidite linker units suitable for use on commercial DNA synthesis machines have been designed for the direct incorporation of biotin and a new reporter group, phosphotyrosine, at multiple sites on synthetic oligonucleotides. The units are based on a 3-carbon glyceryl backbone where the reporter group is attached to the 2-O-position through a 3-aminopropyl spacer. 17-mer oligonucleotides were synthesized carrying at the 5'-end 1, 2, 4 or 8 biotinyl units or 1, 2, 4 or 8 phosphotyrosinyl units respectively and used for the detection of DNA on nitrocellulose filters by hybridization. Subsequent incubation of the filters with a monoclonal antibody to the reporter group followed by secondary detection using enhanced chemiluminescence (ECL) resulted in amplification of signal strengths as the number of reporter groups was increased. The results were quantitated by use of a charge couple device (CCD) camera. Spacing of biotin moieties by thymidyl residues resulted in further improvements in signal strengths, whereas similar spacing of phosphotyrosinyl units did not.

译文

适用于商业DNA合成机器的非核苷亚磷酰胺接头单元已设计用于在合成寡核苷酸的多个位点上直接掺入生物素和新的报告基团磷酸酪氨酸。单元基于3-碳甘油基骨架,其中报告基团通过3-氨基丙基间隔体连接到2-o位。合成了17-聚体寡核苷酸,分别在5 '端携带1、2、4或8个生物素基单元或1、2、4或8个磷酸基酪氨酸基单元,并用于通过杂交检测硝酸纤维素过滤器上的DNA。随后将滤镜与单克隆抗体孵育到报告组,然后使用增强的化学发光 (ECL) 进行二次检测,随着报告组数量的增加,信号强度得到了放大。通过使用电荷耦合器件 (CCD) 相机对结果进行定量。胸苷基残基对生物素部分的间距导致信号强度的进一步提高,而磷酸基酪氨酸单元的间距却没有。

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