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A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

Purhonen, Janne ; Banerjee, Rishi ; McDonald, Allison E. ; Fellman, Vineta LU orcid and Kallijärvi, Jukka LU (2020) In Nucleic Acids Research 48(15). p.87-87
Abstract

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In... (More)

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Nucleic Acids Research
volume
48
issue
15
pages
87 - 87
publisher
Oxford University Press
external identifiers
  • pmid:32573728
  • scopus:85090491815
ISSN
1362-4962
DOI
10.1093/nar/gkaa516
language
English
LU publication?
yes
id
4b686271-6366-4e2c-b3a2-90a8f4b5a334
date added to LUP
2020-09-29 15:06:20
date last changed
2024-03-20 16:01:52
@article{4b686271-6366-4e2c-b3a2-90a8f4b5a334,
  abstract     = {{<p>Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.</p>}},
  author       = {{Purhonen, Janne and Banerjee, Rishi and McDonald, Allison E. and Fellman, Vineta and Kallijärvi, Jukka}},
  issn         = {{1362-4962}},
  language     = {{eng}},
  number       = {{15}},
  pages        = {{87--87}},
  publisher    = {{Oxford University Press}},
  series       = {{Nucleic Acids Research}},
  title        = {{A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase}},
  url          = {{http://dx.doi.org/10.1093/nar/gkaa516}},
  doi          = {{10.1093/nar/gkaa516}},
  volume       = {{48}},
  year         = {{2020}},
}