Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase.
Thymidine analogues bearing a variety of functional groups at the C5-position via an amino-linker arm were prepared and the substrate activity for PCR using thermophilic KOD Dash DNA polymerase was examined. The enzyme accepted the thymidine analogues bearing pyridine, imidazole, biotin, a cationic-charged guanidinium, a cationic-charged amino, mercaptopyridyl and phenanthrolne groups at the C5-position, forming the corresponding PCR product. However, a thymidine analogue bearing a carboxyl group at the C5-position was a poor substrate and the corresponding PCR products could not be obtained. The thymidine analogue bearing a mercapto group was also a poor substrate for the enzyme, because it dimerized by disulfide linkage under PCR conditions. The enzyme hardly accepts the thymidine analogues with a negatively-charged carboxyl group or a bulky group as a substrate. KOD Dash DNA polymerase, having a broader substrate specificity than any other DNA polymerase, will expand the variety of modified DNAs that can be prepared by PCR.
Department of Applied Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan.
Tsutomu
T
Ohbayashi
Masayasu
M
Kuwahara
Masatoshi
M
Hasegawa
Toshiyuki
T
Kasamatsu
Takehiro
T
Tamura
Hiroaki
H
Sawai
D000588:Amines / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000138:chemical synthesis*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011725:Pyridines / Q000737:chemistry; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry*
D000588:Amines; D007093:Imidazoles; D011725:Pyridines; D001710:Biotin; C029899:imidazole; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D019791:Guanidine; C023666:pyridine; D013936:Thymidine