Elimination of residual natural nucleotides from 3′-O-modified-dNTP syntheses by enzymatic mop-up
Elimination of residual natural nucleotides from 3′-O-modified-dNTP syntheses by enzymatic mop-up
复制标题
DOI：
10.2144/98255st01
复制
发表时间：
1998-11-01
期刊：
影响因子：
2.7
通讯作者：
Gibbs, RA
中科院分区：
文献类型：
--
作者：
Metzker, ML;Raghavachari, R;Gibbs, RA
在这里，MLE描述了一种被称为酶“MOP-UP”的新策略，该策略可以有效地从反相、高效液相色谱(RP-HPLC)纯化的3‘-O-修饰的dNTP合成中去除污染的dNTP。酶促清除，利用DNA聚合酶对前一种核苷酸三磷酸的高选择性，而不是后一种核苷酸类似物。我们证明了分别从3‘-O-甲基-dATP和3’-O-(2-硝基苄基)-dTTP合成产物中选择性地去除污染的dATP和dTTP。这些数据强调了在解释酶掺入数据时自然核苷酸污染的重要性，并为观察到的DNA聚合酶催化编辑的性质提供了另一种假说。此外，从3‘-O修饰的类似物中有效地去除天然核苷酸解决了核苷酸读出的重要问题，用于停止-开始DNA测序策略，例如玻色加成测序方案(BASS)。
Here, Mle describe a novel strategy called enzymatic "Mop-Up" that efficiently removes contaminating dNTPs from reverse-phase, high-performance liquid chromatography (RP-HPLC) purified 3'-O-modified dNTP syntheses. Enzymatic mop-up, takes advantage of the high selectivity of DNA polymerases for the former nucleoside triphosphates over the latter nucleotide analogs. We demonstrate the selective removal of contaminating dATP and dTTP from RP-HPLC purified 3'-O-methyl-dATP and 3'-O-(2-nitrobenzyl)-dTTP syntheses, respectively. These data highlight the importance of natural nucleotide contamination when interpreting enzymatic incorporation data and provide an alternative hypothesis for the observed property of catalytic editing of DNA polymerases. Moreover, the effective removal of natural nucleotides from 3'-O-modified analogs addresses the important issue of nucleotide read-through for stop-start DNA sequencing strategies, such as the bose addition sequencing scheme (BASS).