Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.
We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.
Kuwahara
Masayasu
M
Department of Applied Chemistry, Faculty of Engineering, Gunma University, Gunma 376-8515, Japan. kuwahara@chem.gunma-u.ac.jp
Nagashima
Jun-ichi
J
Hasegawa
Masatoshi
M
Tamura
Takehiro
T
Kitagata
Rina
R
Hanawa
Kazuo
K
Hososhima
Shin-ichi
S
Kasamatsu
Toshiyuki
T
Ozaki
Hiroaki
H
Sawai
Hiroaki
H
D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000032:analysis; D010710:Phosphates / Q000737:chemistry; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D017931:DNA Primers; D003845:Deoxycytosine Nucleotides; D003856:Deoxyuracil Nucleotides; D009711:Nucleotides; D010710:Phosphates; C027078:deoxyuridine triphosphate; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase