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Tytuł pozycji:

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

Tytuł:
Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples.
Autorzy:
Ranjbarian F; Dept. Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Sharma S; Dept. Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Falappa G; Dept. Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Taruschio W; Dept. Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Chabes A; Dept. Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Hofer A; Dept. Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Źródło:
Nucleic acids research [Nucleic Acids Res] 2022 Feb 22; Vol. 50 (3), pp. e18.
Typ publikacji:
Journal Article; Research Support, Non-U.S. Gov't
Język:
English
Imprint Name(s):
Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.
MeSH Terms:
Chromatography, High Pressure Liquid*/methods
Deoxyribonucleotides*/analysis
Ribonucleotides/*analysis
Adenosine Diphosphate ; DNA ; Reproducibility of Results
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Substance Nomenclature:
0 (Deoxyribonucleotides)
0 (Ribonucleotides)
61D2G4IYVH (Adenosine Diphosphate)
9007-49-2 (DNA)
Entry Date(s):
Date Created: 20211201 Date Completed: 20220415 Latest Revision: 20220531
Update Code:
20240105
PubMed Central ID:
PMC8860589
DOI:
10.1093/nar/gkab1117
PMID:
34850106
Czasopismo naukowe
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.
(© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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