PCR Incorporation of Modified dNTPs: The Substrate Properties of Biotinylated dNTPs

Paul, Natasha; Yee, Joyclyn

doi:10.2144/000113405

Cited by 21 publications

(20 citation statements)

References 3 publications

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“…We extended the capabilities of single-strand synthesis to incorporate modified dNTPs dispersed throughout the entire polymer for therapeutic and theranostic or imaging applications, similar to what has been shown in dsDNA synthesis 24 , 25 . We tested this strategy using four different dNTPs, replacing in varying percentages one or all four of the canonical dNTPs.…”

Section: Resultsmentioning

confidence: 99%

In vitro synthesis of gene-length single-stranded DNA

Veneziano

Shepherd

Ratanalert

et al. 2018

Sci Rep

111

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Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxicity. However, ssDNA synthesis strategies are limited by the maximum length attainable, ranging from a few hundred nucleotides for chemical synthesis to a few thousand nucleotides for enzymatic synthesis, as well as limited control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kilobases (kb) using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and theranostic applications.

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Section: Resultsmentioning

confidence: 99%

In vitro synthesis of gene-length single-stranded DNA

Veneziano

Shepherd

Ratanalert

et al. 2018

Sci Rep

111

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“… 39 In addition to 5-medCTP, previous studies also demonstrated that many other modified NTPs can be incorporated into DNA and RNA during in vitro replication and transcription. 14 , 16 , 40 – 42 In this respect, these detectable endogenous modified NTPs can be theoretically incorporated into DNA and RNA, which may compromise the regulatory roles of modifications.…”

Section: Resultsmentioning

confidence: 99%

Modified nucleoside triphosphates exist in mammals

et al. 2018

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“…As shown in Figure , increasing the concentration of NH 2 ‐dATP resulted in relatively weak DNA bands, which indicated a lower yield in case of the 1:3 molar ratio. It has been reported that PCR yield is strongly dependent on chemical nature of the modified nucleotide and DNA polymerase used . Therefore, the PCR conditions were determined experimentally.…”

Section: Resultsmentioning

confidence: 99%

The covalent bioconjugate of multiwalled carbon nanotube and amino‐modified linearized plasmid DNA for gene delivery

Geyik

Evran

Timur

et al. 2013

Biotechnology Progress

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Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well-known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The whole pQE-70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6-(6-Amino)hexyl-2'-deoxyadenosine-5'-triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino-modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat-shock step at 42°C. The presence of Ca(2+) in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.

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PCR Incorporation of Modified dNTPs: The Substrate Properties of Biotinylated dNTPs

Cited by 21 publications

References 3 publications

In vitro synthesis of gene-length single-stranded DNA

In vitro synthesis of gene-length single-stranded DNA

Modified nucleoside triphosphates exist in mammals

The covalent bioconjugate of multiwalled carbon nanotube and amino‐modified linearized plasmid DNA for gene delivery

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