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Unnatural nucleotides for DNA sequencing
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Fluorescent nucleotide analogs were prepared and tested to find surrogate structures that are: (i) incorporated by DNA polymerases; (ii) spectroscopically distinct for each fluorescent tag; and (iii) easily deprotected at the 3'-position under mild conditions to reveal the 3'-hydroxy group. These nucleotide analogs are utilized in the Base Addition Sequencing Scheme (BASS) method developed by the Burgess and Gibbs'group. 3'-Blocked nucleotides, 2'-deoxy-3'-O-methylthymidine and 2'-deoxy-3'-Omethylcytidine triphosphates were synthesized and tested as terminators for DNA sequencing. In a Sanger-type DNA sequencing assay, results have shown that 2'-deoxy3'-O-methylthyniidine acted as a terminator against Bst DNA polymerase, AmpliTaq[ ] DNA polymerase, VentR(exo-)@ DNA polymerase and rTth. DNA polymerase. This demonstrates the possible role of 3'-O-methyl-dTTP as an alternative terminator to ddTTP. A fluorescent 3'safety-catch linker nucleoside with a photolabile protecting group was prepared via a convergent synthesis which involved two compounds, Nvoc-Lys(dansyl)OH and H-Pro-ONucleoside. The safety-catch linker was designed to be liberated through a photochemical trigger. Photodecomposition of the fluorescent nucleoside analog was examined using 360 nm irradiation. Without any additives, 5'-silylated thymidine was not formed, but the free amine intermediate accumulated instead. In the presence of ethanolamine, however, 5'-silylated thymidine formed at a rate which increases with the concentration of ethanolamine. Syntheses of 3-nitropyffole and 5-nitroindole nucleotides were performed. These modified nucleotides were tested as substrates of several commercially available DNA polymerases. They were utilized in the assay as viable substitutes of DATP. Results have shown that 3-nitropyrrole nucleotide is a potential surrogate for DATP. However, 5 nitroindole nucleotide showed mostly termination activity against several commercially available DNA polymerases. The behavior of 3-nitropyrrole nucleotide under MALDI-MS conditions was also investigated. Phosphoramidite analog of 3-nitropyr-role nucleoside was prepared and incorporated into the oligonucleotide dT4XT5 using an ABI DNA synthesizer. Oligonucleotide dT4AT5 was also prepared for comparison. MALDI-MS analyses revealed that the oligonucleotide containing 3-nitropyrrole nucleoside is less prone to fragmentation than dT4AT5. This shows that 3-nitropyrrole is a superior base compared to adenosine for MALDI-MS DNA sequencing purposes.
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Jacutin, Swanee E (1997). Unnatural nucleotides for DNA sequencing. Master's thesis, Texas A&M University. Available electronically from
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