Direct PCR amplification of various modified DNAs having amino acids: convenient preparation of DNA libraries with high-potential activities for in vitro selection.
We synthesized modified 2'-deoxyuridine triphosphates bearing amino acids at the C5 position and investigated their substrate properties for KOD Dash DNA polymerase during polymerase chain reaction (PCR). PCR using C5-modified dUTP having an amino acyl group (arginyl, histidyl, lysyl, phenylalanyl, tryptophanyl, leucyl, prolyl, glutaminyl, seryl, O-benzyl seryl or threonyl group) gave the corresponding full-length PCR products in good yield. Although dUTP analogues bearing aspartyl, glutamyl or cysteinyl were found to be poor substrates for PCR catalyzed by KOD Dash DNA polymerase, optimization of the reaction conditions resulted in substantial generation of full-length product. In the case of reaction using dUTP analogue having a cysteinyl group, addition of a reducing agent improved the reaction yield. Thus, PCRs using KOD Dash DNA polymerase together with amino acyl dUTP provide convenient and efficient preparation of various modified DNA libraries with potential protein-like activities.
Kuwahara
Masayasu
M
Department of Applied Chemistry, Faculty of Engineering, Gunma University, Gunma 376-8515, Japan.
Hanawa
Kazuo
K
Ohsawa
Kazuomi
K
Kitagata
Rina
R
Ozaki
Hiroaki
H
Sawai
Hiroaki
H
D000596:Amino Acids / Q000737:chemistry*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D000596:Amino Acids; D017931:DNA Primers; D004247:DNA