Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates

Molecules

Erkai Liu²  Curtis H. Lam²  David M. Perrin¹ 

[1]Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T1Z1, Canada

关键词: modified nucleoside triphosphates;    Vent (exo-) DNA polymerase;    incorporation;   

DOI  :  10.3390/molecules200813591

来源: mdpi

【 摘 要 】

To expand the chemical functionality of DNAzymes and aptamers, several new modified deoxyuridine triphosphates have been synthesized. An important precursor that enables this aim is 5-aminomethyl dUTP, whereby the pendent amine serves as a handle for further synthetic functionalization. Five functional groups were conjugated to 5-aminomethyl dUTP. Incorporation assays were performed on several templates that demand 2–5 sequential incorporation events using several commercially available DNA polymerases. It was found that Vent (exo-) DNA polymerase efficiently incorporates all five modified dUTPs. In addition, all nucleoside triphosphates were capable of supporting a double-stranded exponential PCR amplification. Modified PCR amplicons were PCR amplified into unmodified DNA and sequenced to verify that genetic information was conserved through incorporation, amplification, and reamplification. Overall these modified dUTPs represent new candidate substrates for use in selections using modified nucleotide libraries.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.