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

:已设计出适用于商业DNA合成机的非核苷亚磷酰胺连接体单元,用于在合成寡核苷酸的多个位点直接掺入生物素和新的报告基团磷酸酪氨酸。这些单元基于3-碳甘油基主链,其中报告基团通过3-氨丙基间隔基连接到2-O-位。合成分别具有5'端1、2、4或8个生物素基单元或1、2、4或8个磷酸酪氨酸基单元的17聚体寡核苷酸,并用于通过杂交在硝酸纤维素滤膜上检测DNA。随后将滤膜与针对报告基因组的单克隆抗体一起孵育,然后使用增强的化学发光(ECL)进行二次检测,导致信号强度随报告基因组数量的增加而放大。通过使用电荷耦合器件(CCD)相机对结果进行定量。胸苷残基对生物素部分的间隔导致信号强度的进一步提高,而磷酸酪氨酰基单元的相似间隔却没有。

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