TY - JOUR
T1 - Assay of deoxy-deazapurines in DNA by 3'-phosphorylation and two-dimensional thin-layer chromatography
AU - Steinberg, J. J.
AU - Oliver, Gary W.
AU - Cajigas, Antonio
N1 - Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1993/2/26
Y1 - 1993/2/26
N2 - Deoxy-deazapurines (deaza-dNMPs) are incorporated into cellular DNA after administration of anti-neoplastic, anti-viral, or anti-parasitic chemotherapy. Deaza-dNMPs are stable purine analogues and can be detected via 32P-labeling cold DNA. Assay of analogue incorporation and normal base composition is carried out by radiolabeling DNA with all four deoxynucleotides (dNMPs) through nick translation. 3'-Monophosphate digest radiolabels representative dNMPs and deaza-dNMPs. Separation occurs in two-dimensional polyethyleneimine-cellulose thin-layer chromatography, which resolves all dNMPs. The technique was applied to human placental and calf thymus DNA, control and altered calf thymus DNA with cold stoichiometric replacement of deaza-dNMPs to include deoxy-deazaadenosine, deoxy-deazaguanosine, and deoxy-deazainosine. Scintillation detection and densitometry both accurately reflect dNMP content. This technique easily and quickly quantifies the low-molecular-mass deaza-dNMP analogues in DNA. Deaza-dNMP uptake into DNA may reflect clinical chemotherapeutic efficacy and host toxicity. The assay may therefore serve as an early biochemical dosimeter of drug effect and resistance.
AB - Deoxy-deazapurines (deaza-dNMPs) are incorporated into cellular DNA after administration of anti-neoplastic, anti-viral, or anti-parasitic chemotherapy. Deaza-dNMPs are stable purine analogues and can be detected via 32P-labeling cold DNA. Assay of analogue incorporation and normal base composition is carried out by radiolabeling DNA with all four deoxynucleotides (dNMPs) through nick translation. 3'-Monophosphate digest radiolabels representative dNMPs and deaza-dNMPs. Separation occurs in two-dimensional polyethyleneimine-cellulose thin-layer chromatography, which resolves all dNMPs. The technique was applied to human placental and calf thymus DNA, control and altered calf thymus DNA with cold stoichiometric replacement of deaza-dNMPs to include deoxy-deazaadenosine, deoxy-deazaguanosine, and deoxy-deazainosine. Scintillation detection and densitometry both accurately reflect dNMP content. This technique easily and quickly quantifies the low-molecular-mass deaza-dNMP analogues in DNA. Deaza-dNMP uptake into DNA may reflect clinical chemotherapeutic efficacy and host toxicity. The assay may therefore serve as an early biochemical dosimeter of drug effect and resistance.
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U2 - 10.1016/0378-4347(93)80174-3
DO - 10.1016/0378-4347(93)80174-3
M3 - Article
C2 - 8468386
AN - SCOPUS:0027405857
SN - 0378-4347
VL - 612
SP - 277
EP - 285
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
IS - 2
ER -