| Budget Amount *help |
¥14,500,000 (Direct Cost: ¥14,500,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2002: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2001: ¥7,900,000 (Direct Cost: ¥7,900,000)
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| Research Abstract |
We prepared various novel C5-substituted 2'-deoxyuridine-5'-triphosphates, which have an amino group, a carboxyl group, imidazole derivatives, pyridine, biotin, chelating groups, fluorescence and amino acids at a C5-terminal via a linker. Many of them could work as a substrate for several thermophilic DNA polymerases. B-Family DNA polymerases obtained from thermophilic archaeas, such as KOD dash, Vent(exo-) and Pwo DNA polymerase, could accept the C5-substituted deoxyuridine derivatives and gave the corresponding modified DNA by PCR. On the other hand, A-family DNA polymerases obtained from thermophilic bacteria, such as Taq DNA polymerase, could not accept these modified 2'-deoxyuridine derivatives at all. The C5 substituted deoxyuridine derivatives bearing an anionic carboxyl group, a bulky group with a short linker or sulfur-containing compounds were a poor substrate for any DNA polymerases. Post-synthetic functionalization of the modified DNA obtained by PCR extends the varieties of the resulting modified DNA. We also prepared 2'-deoxycytidine derivatives bearing several functional groups at C5-position and assessed their substrate properties for the thermophilic DNA polymerases. KOD dash and Vent(exo-) DNA polymerases could accept some C5-substituted 2'-deoxycytidines forming the cytidine-modified DNA by PCR. Double-modified DNA containing the modified 2'-deoxyuridine and modified 2'-deoxycytidine derivatives could be prepared enzymatically by using KOD dash or Vent(exo-) DNA polymerase. We applied the modified DNA synthesis by PCR for the production of a new modified DNA aptamer and a modified DNA catalyst by combination with in vitro selection method. The modified DNA aptamers, which bind with sialyllactose, thalidomide or aspartame were produced, and their binding abilities and sequences were examined to asses the roles of the functional groups in the modified DNA for the binding.
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