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Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates Liu, Erkai; Lam, Curtis H.; Perrin, David M.

Abstract

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.

Item Metadata

Title
Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates
Creator
Liu, Erkai; Lam, Curtis H.; Perrin, David M.
Publisher
Multidisciplinary Digital Publishing Institute
Date Issued
2015-07-24
Description
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.
Subject
Modified nucleoside triphosphates; Vent (exo-) DNA polymerase; Incorporation
Genre
Article
Type
Text
Language
eng
Date Available
2019-05-22
Provider
Vancouver : University of British Columbia Library
Rights
CC BY 4.0
DOI
10.14288/1.0378915
URI
http://hdl.handle.net/2429/70279
Affiliation
Science, Faculty of; Chemistry, Department of
Citation
Molecules 20 (8): 13591-13602 (2015)
Publisher DOI
10.3390/molecules200813591
Peer Review Status
Reviewed
Scholarly Level
Faculty
Rights URI
https://creativecommons.org/licenses/by/4.0/
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CC BY 4.0