Conventional pyrosequencing using 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPαS) is problematic due to the high cost of the substrate (dATPαS) and deterioration in the accuracy of incorporation to read through poly(T) regions. One reason for these problems is that dATPαS has a sulfur on the α-phosphate and also has isomers (Sp and Rp). To solve these problems, 11 nucleotide substrates, which could replace dATPαS in pyrosequencing, were newly synthesized. All substrates were modified on the seventh or eighth position of the adenine base from normal dATP. We found that the substrate that had an ethenyl-linked modified group on the seventh position of the adenine base had low activity in the luciferase reaction and high incorporation efficiency with the thymine base. One substrate in particular had 10-fold better incorporation efficiency than dATPαS. The new nucleotide substrate satisfied all conditions as a replacement of dATPαS.
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