Metzker M L, Raghavachari R, Burgess K, Gibbs R A
Baylor College of Medicine, Houston, TX, USA.
Biotechniques. 1998 Nov;25(5):814-7. doi: 10.2144/98255st01.
Here, we 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 base addition sequencing scheme (BASS).
在此，我们描述了一种名为酶促“清除”的新策略，该策略能有效从反相高效液相色谱（RP-HPLC）纯化的3'-O-修饰的dNTP合成物中去除污染的dNTP。酶促清除利用了DNA聚合酶对前者核苷三磷酸比对后者核苷酸类似物的高选择性。我们分别展示了从RP-HPLC纯化的3'-O-甲基-dATP和3'-O-(2-硝基苄基)-dTTP合成物中选择性去除污染的dATP和dTTP。这些数据突出了在解释酶促掺入数据时天然核苷酸污染的重要性，并为观察到的DNA聚合酶催化编辑特性提供了另一种假设。此外，从3'-O-修饰的类似物中有效去除天然核苷酸解决了用于起止DNA测序策略（如碱基添加测序方案（BASS））的核苷酸通读这一重要问题。
