Enzymatic incorporation of chemically-modified nucleotides into DNAs.
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.
Kuwahara
Masayasu
M
Department of Chemistry, Gunma University, 1-5-1 Tenjin-chou, Kiryu, Gunma 376-8515, Japan.
Ohbayashi
Tsutomu
T
Hanawa
Kazuo
K
Shoji
Atsushi
A
Ozaki
Akiko N
AN
Ozaki
Hiroaki
H
Sawai
Hiroaki
H
D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction
D009711:Nucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase