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Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples

Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An...

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Autores principales: Ranjbarian, Farahnaz, Sharma, Sushma, Falappa, Giulia, Taruschio, Walter, Chabes, Andrei, Hofer, Anders
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8860589/
https://www.ncbi.nlm.nih.gov/pubmed/34850106
http://dx.doi.org/10.1093/nar/gkab1117
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author Ranjbarian, Farahnaz
Sharma, Sushma
Falappa, Giulia
Taruschio, Walter
Chabes, Andrei
Hofer, Anders
author_facet Ranjbarian, Farahnaz
Sharma, Sushma
Falappa, Giulia
Taruschio, Walter
Chabes, Andrei
Hofer, Anders
author_sort Ranjbarian, Farahnaz
collection PubMed
description Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.
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spelling pubmed-88605892022-02-22 Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples Ranjbarian, Farahnaz Sharma, Sushma Falappa, Giulia Taruschio, Walter Chabes, Andrei Hofer, Anders Nucleic Acids Res Methods Online Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection. Oxford University Press 2021-11-25 /pmc/articles/PMC8860589/ /pubmed/34850106 http://dx.doi.org/10.1093/nar/gkab1117 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods Online
Ranjbarian, Farahnaz
Sharma, Sushma
Falappa, Giulia
Taruschio, Walter
Chabes, Andrei
Hofer, Anders
Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples
title Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples
title_full Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples
title_fullStr Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples
title_full_unstemmed Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples
title_short Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples
title_sort isocratic hplc analysis for the simultaneous determination of dntps, rntps and adp in biological samples
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8860589/
https://www.ncbi.nlm.nih.gov/pubmed/34850106
http://dx.doi.org/10.1093/nar/gkab1117
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