Kuwahara Masayasu, Ohbayashi Tsutomu, Hanawa Kazuo, Shoji Atsushi, Ozaki Akiko N, Ozaki Hiroaki, Sawai Hiroaki
Department of Chemistry, Gunma University, 1-5-1 Tenjin-chou, Kiryu, Gunma 376-8515, Japan.
Nucleic Acids Res Suppl. 2002(2):83-4. doi: 10.1093/nass/2.1.83.
Modified 2'-deoxyuridine triphosphates bearing proteinous amino acids at a C5 position were prepared, and their substrate properties were investigated for KOD Dash DNA polymerase during PCR. The modified dUTPs bearing histidyl, lysyl, glutaminyl or seryl group produced the aimed 108 nt PCR products in good yields. In contrast, the analog bearing glutamyl group did not work as a substrate for KOD Dash while the analog bearing aspartyl group gave the product in a low yield. Moreover, not only KOD Dash but also three other thermostable DNA polymerases were tested as catalysts by use of C5 modified dUTPs with two different types of linker arms. Both Pfu and Vent(exo-) were relatively tolerant for the modification at the C5 position as well as KOD Dash.
制备了在C5位带有蛋白质氨基酸的修饰2'-脱氧尿苷三磷酸，并在PCR过程中研究了它们作为KOD Dash DNA聚合酶底物的性质。带有组氨酸、赖氨酸、谷氨酰胺或丝氨酸基团的修饰dUTP以良好的产率产生了目标108 nt的PCR产物。相比之下，带有谷氨酰基的类似物不能作为KOD Dash的底物，而带有天冬氨酰基的类似物产率较低。此外，不仅使用了KOD Dash，还使用带有两种不同类型连接臂的C5修饰dUTP测试了另外三种热稳定DNA聚合酶作为催化剂。Pfu和Vent(exo-)与KOD Dash一样，对C5位的修饰相对耐受。
