Enzymatic Synthesis of Labeled DNA by PCR Using New Fluorescent Thymidine Nucleotide Analogue and Superthermophilic KOD Dash DNA Polymerase Tsutomu Obayashi, Mohammed M. Masud, Akiko N. Ozaki, Hiroaki Ozaki, Masayasu Kuwahara and Hiroaki Sawai* Department of Applied Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan Received 19 December 2001; accepted 9 February 2002 Abstract—Triphosphate of a new fluorescent labeled thymidine analogue was incorporated as a substrate for PCR using KOD Dash DNA polymerase forming the corresponding fluorescent labeled DNA which is useful for a DNA probe. # 2002 Elsevier Science Ltd. All rights reserved. Fluorescence-labeled DNA probes are important tools for biological and biochemical studies and for diag- nostic use. A variety of methods for the synthesis of the fluorescent DNA have been reported over the past 10 years. 1 3 Probes containing higher density of labels would increase detection sensitivity. Polymeric chain reaction is very useful for the simultaneous labeling and the amplification of DNA with high density of the fluorophore, if a fluorescent nucleotide can be accepted by DNA polymerase as a substrate. It is reported that Taq and Vent DNA polymerases can use 5 0 -triphos- phates of 2 0 -deoxyuridine derivatives bearing a (E)-pro- penyl or propynyl linker at C5 position, but no other modified substrate analogue. 7 11 Thus, 2 0 -deoxyuridine derivatives bearing a fluorophore via 3-aminopropenyl or 3-aminopropynyl linker at C5 position have been developed and used as a fluorescent-labeled substrate for Taq DNA polymerase, although co-existence of the natural substrate TTP is required for the synthesis of a full-length DNA probe. 3 6 For example, Ried et al. reported synthesis and application of the DNA probes by PCR using Taq DNA polymerase and the fluore- sence-labeled deoxyuridine derivative with a 3-amino- propenyl linker. 4 Cyanine dye-labeled deoxyuridine derivatives with a 3-aminopropenyl linker can be also accepted by Taq DNA polymerase yielding the corre- sponding fluorescent labeled DNA probe. 5,6 Although they are now commercially available, more efficient labeled nucleotides and enzymes are required to prepare the labeled DNA probes for use in the diagnosis and in the molecular biology. Previously, we reported the synthesis of thymidine ana- logues bearing a methylene group at the C5 a-position with an amino-linker arm, and their introduction into oligodeoxyribonucleotides 12,13 using a DNA synthesi- zer. Very recently, we reported that triphosphates of the thymidine analogues could be accepted by KOD Dash DNA polymerase as a substrate in the PCR. 14 The tri- phosphate of the thymidine analogue with amino-linker was further reacted with fluorescein isothiocyanate, yielding a new fluorescence-labeled thymidine analogue. Here we report preparation of the fluorescent nucleo- tides and their application for the synthesis of fluores- cence-labeled DNA by PCR using KOD Dash DNA polymerase. Post-labeling of the modified DNA pre- pared from the thymidine analogue by PCR was also carried out by reaction with fluorescein isothiocyanate. 5 0 -Triphosphate of 5 - N - (6 - aminohexyl)carbamoyl- methyl-2 0 -deoxyuridine (1) was synthesized by the method described previously. 14,15 The terminal amino group of the nucleotide was reacted with 4.4 equivalent of fluorescein isothiocyanate in 0.25 M carbonate buffer (pH 9.2) containing DMF to furnish the fluorescence labeled-thymidine analogue (2) in 28% yield, after purification by HPLC. The nucleotide 1 was reacted with N-[e-trifluoroacetylcaproyloxy]succinimide ester, 0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(02)00111-7 Bioorganic & Medicinal Chemistry Letters 12 (2002) 1167–1170 *Corresponding author. Tel.: +81-277-30-1220; fax: +81-277-30- 1224; e-mail: sawai@chem.gunma-u.ac.jp