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Org. Biomol. Chem., 2005, 3, 2463 - 2468, DOI: 10.1039/b504330a
Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase
Tsutomu Ohbayashi, Masayasu Kuwahara, Masatoshi Hasegawa, Toshiyuki Kasamatsu, Takehiro Tamura and Hiroaki Sawai
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.
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